ISOLATED ANTIBODY OR ITS IMMUNORREATIVE FRAGMENT, NUCLEIC ACID MOLECULE, PHARMACEUTICAL COMPOSITION,

专利摘要:
isolated antibody or its immunoreactive fragment, hybridoma, nucleic acid molecule, dual affinity (dartth) redirection reagent, pharmaceutical composition, and, use of isolated antibody the present invention relates to antibodies and fragments thereof that are immunoreactive to mammals, and more particularly, the human b7-h3 receptor and their use, particularly in the treatment of cancer and inflammation. the invention thus particularly relates to reactive b7-h3 humanized antibodies and immunoreactive fragments thereof which are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells which are associated with a variety of human cancers;
公开号:BR112012022210B1
申请号:R112012022210-4
申请日:2011-03-01
公开日:2021-08-17
发明作者:Deryk T. Loo；Ling Huang；Leslie S. Johnson；Francine Zhifen Chen；Paul A. Moore
申请人:Macrogenics, Inc；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS:
[001] This patent application claims priority to the Serial Numbers of US Patent Applications 61/310. 692 (filed March 4, 2010; pending); 61/310,695 (filed on March 4, 2010; pending) 61/311. 057 (filed March 5, 2010; pending), each such patent application is incorporated herein by reference in its entirety. REFERENCE TO THE SEQUENCE LISTING:
[002] This patent application includes one or more Sequence Listings pursuant to C.F.R. 37 1,821 et seq., which are described in both paper and computer readable media, and whose paper and computer readable descriptions are incorporated herein by reference in their entirety. HISTORY OF THE INVENTION: FIELD OF THE INVENTION
[003] The present invention relates to antibodies and fragments thereof that are immunoreactive to the mammal, and more particularly, to the human B7-H3 receptor and their uses, particularly in the treatment of cancer and inflammation. The invention thus particularly relates to humanized antibodies reactive to B7-H3 and its immunoreactive fragments that are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells that are associated with a variety of human cancers. DESCRIPTION OF RELATED MATTER:
[004] The growth and metastasis of tumors depend to a large extent on their ability to evade host immune surveillance and overcome host defenses. Most tumors express antigens that can be recognized to a variable extent by the host's immune system, but in many cases, an inadequate immune response is elicited because of ineffective activation of effector T cells (Khawli, LA et al. (2008) ) "Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors," Exper. Pharmacol. 181:291-328).
[005] CD4+ T lymphocytes are the essential organizers of most mammalian immune and autoimmune responses (Dong, C. et al. (2003) "Immune Regulation by Novel Costimulatory Molecules" Immunolog. Res. 28(l):39- 48). Activation of CD4+ helper T cells was found to be mediated through costimulatory interactions between antigen-presenting cells and natural CD4+ T lymphocytes. Two interactions are required (Viglietta, V. et al. (2007) "Modulating Co-Stimulation" Neurotherapeutics 4:666-675; Korman, AJ et al. (2007) "Checkpoint Blockade in Cancer Immunotherapy" Adv. Immunol. 90: 297-339). In the first interaction, an Antigen Presenting Cell must display the relevant target antigen bound to the cell's major histocompatibility complex so that it can bind to the T cell receptor (“TCR”) of a natural CD4+ T lymphocyte. In the second interaction, an antigen-presenting cell ligand must bind to a CD28 receptor on the CD4+ T lymphocyte (Dong, C. et al. (2003) “Immune Regulation by Novel Costimulatory Molecules,” Immunolog. Res. 28(l) :39-48; Lindley, PS et al. (2009) "The Clinical Utility Of Inhibiting CD28-Mediated Costimulation," Immunol. Rev. 229:307-321). CD4+ helper T cells that experience both stimulatory signals are then able to respond to cytokines (such as Interleukin-2 and Interleukin-12 to develop into Th1 cells). Such cells produce interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TNF-α), which mediate inflammatory responses to target cells that express the target antigen. B cell activation and proliferation also occur, resulting in the production of antibody specific for the target antigen (Bernard, A. et al. (2005) "T and B Cell Cooperation: A Dance of Life and Death" Transplantation 79:S8- S11). In the absence of both costimulatory signals during TCR involvement, T cells enter a functionally unresponsive state, referred to as clonal anergy (Khawli, LA et al. (2008) “Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors,” Exper. Pharmacol. 181:291-328). In disease states, Th1 cells are the protagonists of several organ-specific autoimmune diseases, such as type I diabetes, rheumatoid arthritis, and multiple sclerosis (Dong, C. et al. (2003) “Immune Regulation by Novel Costimulatory Molecules,” Immunolog. Res. 28(1):39-48). I. THE SUPERFAMILY B7 AND B7—H3
[006] Investigations into CD28 receptor ligands led to the characterization of a set of related molecules known as the B7 superfamily (Coyle, AJ et al. (2001) " Nature Immunol. 2(3):203-209; Sharpe, AH et al. (2002) "The B7-CD28 Superfamily" Nature Rev. Immunol. 2:116-126; Greenwald, RJ et al. (2005) " The B7 Family Revisited,” Ann. Rev. Immunol. 23:515-548; Collins, M. et al. (2005) “The B7 Family Of Immune-Regulatory Ligands,” Genome Biol. 6:223.1-223.7; Loke, P. et al (2004) “Emerging Mechanisms of Immune Regulation: The Extended B7 Family and Regulatory T Cells.” Arthritis Res. Ther. 6:208-214; Korman, AJ et al. (2007) “Checkpoint Blockade in Cancer Immunotherapy," Adv. Immunol. 90:297-339; Flies, DB et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity," J. Immunother. 30(3):251-260; Agarwal , A. et al. (2008) “The Role Of Positive Cos timulatory Molecules In Transplantation And Tolerance” Curr. Opinion Organ Transplant. 13:366-372; Lenschow, D.J. et al. (1996) "CD28/B7 System of T Cell Costimulation" Ann. Rev. Immunol. 14:233-258; Wang, S. et al. (2004) “Co-Signaling Molecules Of The B7-CD28 Family In Positive And Negative Regulation Of T Lymphocyte Responses,” Microbes Infect. 6:759-766). Currently, there are seven known members of the family: B7.1 (CD80), B7.2 (CD86), the inducible costimulatory ligand (ICOS-L), the programmed death ligand 1 (PD-L1), the death ligand 2 programmed (PD-L2), B7-H3 and B7-H4 (Collins, M. et al. (2005) "The B7 Family of Immune-Regulatory Ligands," Genome Biol. 6:223,1-223.7).
Members of the B7 family are members of the immunoglobulin superfamily with an immunoglobulin V-like and an immunoglobulin C-like domain (eg, IgV-IgC) (Sharpe, AH et al. (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2:116-126). The IgV and IgC domains of the B7 family members are each encoded by individual exons, with additional exons encoding the leader sequences, transmembrane and cytoplasmic domains. The cytoplasmic domains are short, ranging in length from 19 to 62 amino acid residues, and can be encoded by multiple exons (Collins, M. et al. (2005) "The B7 Family Of Immune-Regulatory Ligands" Genome Biol. 6:223 , 1-223.7). B7-H3 is the simple one in that the main human form contains two parallel extracellular IgV-IgC domains (ie, IgV-IgC-IgV-IgC) (Collins, M. et al. (2005) “The B7 Family Of Immune- Regulatory Ligands,” Genome Biol. 6:223,1-223,7). Members of the B7 family are predicted to form non-covalent, back-to-back type homodimers on the cell surface, and such dimers have been found with respect to B7-1 (CD80) and B7-2 (CD86).
[008] The display of B7-1 (CD80) and B7-2 (CD86) has dual specificity for the stimulatory CD28 receptor and the inhibitory CTLA-4 (CD 152) receptor (Sharpe, AH et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126).
[009] Although initially thought to comprise only 2 Ig domains (IgV-IgC) (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-y Production," Nature Immunol. 2:269-274; Sun, M. et al (2002) "Characterization of Mouse and Human B7-H3 Genes," J. Immunol. 168:6294-6297), a four immunoglobulin extracellular domain variable ("4Ig" -B7-H3”) has been identified and found to be the most common human form of the protein (Sharpe, AH et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126). No functional differences were observed between these two forms, as the natural murine form (2Ig) and the human form of 4Ig exhibit similar function (Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7-H3,” Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278). The 4Ig-B7-H3 molecule inhibits natural killer cell-mediated lysis of cancer cells (Castriconi, R. et al. "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell- Mediated Lysis,” Proc. Natl. Acad. Sci. (USA) 101 (34): 12640-12645). Human B7-H3 (2Ig form) has been found to promote T cell activation and IFN-Y production by binding to a putative receptor on activated T cells (Chapoval, A. et al. (2001) “B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-y Production" Nature Immunol. 2:269-274; Xu, H. et al. (2009) "MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors,” Cancer Res. 69(15):5275-6281). Both B7-H4 and B7-H1 are potent inhibitors of immune function when expressed in tumor cells (Flies, DB et al. (2007) "The New B7s: Playing a Pivotal Role in Tumor Immunity," J. Immunother. 30(3 ):251-260).
[0010] The mode of action of B7-H3 is complex, as the protein mediates both co-stimulation and co-inhibition of the T cell (Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7-H3,” Proc Natl. Acad. Sci. (USA) 105(30):10277-10278; Martin-Orozco, N. et al. (2007) "Inhibitory Costimulation And Anti-Tumor Immunity" Semin. Cancer Biol. 17(4): 288-298; Subudhi, SK et al. (2005) "The Balance Of Immune Responses: Costimulation Verse Coinhibition," J. Mol. Med. 83:193-202). B7-H3 binds to transcript type 2 (TREM) (TLT-2) and co-stimulates T cell activation, but also binds to unidentified receptor(s) to mediate T cell co-inhibition. , B7-H3, through interactions with unknown receptor(s), is an inhibitor for natural killer cells and osteoblastic cells (Hofmeyer, K. et al. (2008) "The Contrasting Role Of B7-H3" Proc. Natl. Acad Sci. (USA) 105(30):10277-10278). Inhibition may operate through interactions with members of the major signaling pathways through which the T cell receptor (TCR) regulates gene transcription (eg, NFTA, NF-KB, or AP-1) factors.
[0011] B7-H3 co-stimulates CD4+ and CD8+ T cell proliferation. B7-H3 also stimulates IFN-Y production and CD8+ lytic activity (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-y Production," Nature Immunol. 2 :269-274; Sharpe, AH et al (2002) "The B7-CD28 Superfamily," Nature Rev. Immunol. 2:116-126). However, the protein also possibly acts through factors NFAT (nuclear factor for activated T cells), NF-kB (nuclear factor kappa B), and AP-1 (Activating Protein-1) to inhibit T cell activation (Yi. KH et al (2009) “Fine Tuning The Immune Response Through B7-H3 And B7-H4,” Immunol. Rev. 229:145-151). B7-H3 is also believed to inhibit Th1, Th2, or Th17 in vivo ( Prasad, DV et al. (2004) "Murine B7-H3 Is A Negative Regulator Of T Cells" J. Immunol. 173:2500-2506; Fukushima, A. et al. (2007) "B7-H3 Regulates The Development Of Experimental Allergic Conjunctivitis In Mice," Immunol. Lett. 113:52-57; Yi. KH et al. (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4," Immunol. Rev. 229:145-151). Several independent studies showed that human malignant tumor cells exhibit a marked increase in B7-H3 protein expression and that this increased expression was associated with increased disease severity (Zang, X. et al. (2007) “The B7 Family And Cancer Therapy : Costimulation And Coinhibition” Clin. Cancer Res. 13:5271-5279), suggesting that B7-H3 is exploited by tumors as an immune evasion pathway (Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7 -H3" Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).
[0012] Molecules that block the ability of a B7 molecule to bind to a T cell receptor (eg, CD28) inhibit the immune system and have been proposed as treatments for autoimmune disease (Linsley, PS et al. (2009) “The Clinical Utility Of Inhibiting CD28-Mediated Co-Stimulation,” Immunolog. Rev. 229:307-321). Neuroblastoma cells expressing 4Ig-B7-H3 treated with anti-4Ig-B7-H3 antibodies were more susceptible to NK cells. However, it is unclear whether this activity can be attributed to only antibodies against the 4Ig-B7-H3 form because all reported antibodies produced against 4Ig-B7-H3 also bind to the Ig-like form of B7H3 (Steinberger, P. et al. al (2004) "Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains," J. Immunol. 172(4): 2352-2359 and Castriconi et al. (2004) "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis," Proc. Natl. Acad. Sci. (USA) 101(34): 12640-12645).
[0013] B7-H3 is not expressed in resting B or T cells, monocytes, or dendritic cells, but is induced in dendritic cells by IFN-Y and in monocytes by GM-CSF (Sharpe, AH et al. (2002) “The B7-CD28 Superfamily,” Nature Rev. Immunol. 2:116-126). The receptor(s) that bind(s) to B7-H3 have not been fully characterized. Recent work has suggested that a receptor would need to be rapidly and transiently regulated on T cells after activation (Loke, P. et al. (2004) "Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T Cells." Arthritis Res. Ther 6:208-214). Recently, the type 2 transcript receptor (TREM) (TLT-2, or TREML2) (King, RG et al. (2006) “Trem-Like Transcript 2 Is Expressed On Cells Of The Myeloid/Granuloid And B Lymphoid Lineage And Is Up-Regulated In Response To Inflammation," J. Immunol. 176:6012-6021; Klesney-Tait, J. et al. (2006) "The TRAIN Receptor Family And Signal Integration," Nat. Immunol. 7:1266-1273 ; Yi. KH et al (2009) "Fine Tuning The Immune Response Through B7-H3 And B7-H4," Immunol. Rev. 229:145-151, which is expressed in myeloid cells, has been shown to be able to bind to B7-H3, and thereby co-stimulate the activation of CD8+ T cells in particular (Zang, X. et al. (2003) "B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation," Proc. Natl. Acad Sci. (USA) 100:10388-10392; Hashiguchi, M. et al. (2008) "Triggering Receptor Expressed On Myeloid Cell-Like Transcript 2 (TLT-2) Is A Counter-Receptor For B7-H3 And Enhances T Cell Responses" Proc. Natl. Acad. Sci. (USA) 105(30):10495- 10500; Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7-H3” Proc. Natl. Academic Sci. (U.S.A.) 105(30):10277-10278).
[0014] In addition to its expression in neuroblastoma cells, human B7-H3 is also known to be expressed in a variety of other cancer cells (eg, non-small cell lung, ovarian and gastric cancers). B7-H3 protein expression has been immunohistologically detected in tumor cell lines (Chapoval, A. et al. (2001) "B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-y Production," Nature Immunol. 2:269-274; Saatian, B. et al (2004) "Expression Of Genes For B7-H3 And Other T Cell Ligands By Nasal Epithelial Cells During Differentiation And Activation," Amer. J. Physiol. Lung Cell. Mol. Physiol 287:L217-L225; Castriconi et al (2004) "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis," Proc. Natl. Acad. Sci (USA) 101(34):12640-12645); Sun, M. et al. (2002) “Characterization of Mouse and Human B7-H3 Genes,” J. Immunol. 168:6294-6297). mRNA expression has been discovered in heart, kidney, lung, liver, pancreas, prostate, colon and osteoblast cells (Collins, M. et al. (2005) “The B7 Family Of Immune-Regulatory Ligands,” Genome Biol. 6 :223.1-223.7). At the protein level, B7-H3 is found in the liver, lung, bladder, testes, prostate, breast, human placenta, and lymphoid organs (Hofmeyer, K. et al. (2008) “The Contrasting Role Of B7-H3” Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278). II. THERAPEUTIC ANTIBODIES
[0015] In addition to their known diagnostic uses, antibodies have been shown to be useful as therapeutic agents. For example, immunotherapy, or the use of antibodies for therapeutic purposes, has been used in recent years to treat cancer. Passive immunotherapy involves the use of monoclonal antibodies in cancer treatments (see, for example, DEVITA, HELLMAN, AND ROSENBERG CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY, EIGHTH EDITION (2008), DeVita, V. et al. Eds., Lippincott Williams & Wilkins, Philadelphia, PA, pp. 537-547, 2979-2990). These antibodies may have inherent biological therapeutic activity either by directly inhibiting tumor cell growth or survival or by their ability to recruit the natural cell-killing activity of the body's immune system. These agents can be administered alone or in conjunction with radiation or therapeutic agents. Rituximab and Trastuzumab, approved for the treatment of non-Hodgkin's lymphoma and breast cancer, respectively, are examples of such therapeutics. Alternatively, the antibodies can be used to make antibody conjugates in which the antibody binds to a toxic agent and targets that agent to the tumor by specifically binding to the tumor. Gemtuzumab ozogamycin is an example of an approved antibody conjugate used to treat leukemia.
[0016] Monoclonal antibodies that bind to cancer cells and have potential uses for diagnosis and therapy have been described (see, for example, the following patent applications that describe, among others, some molecular weights of target proteins: North Patent - U.S. Patent No. 6,054,561 (200 kD c-erbB-2 (Her2), and other unknown antigens from 40 to 200 kD in size) and U.S. Patent No. 5,656,444 (50 kD and 55 kD oncofetal protein) ). Examples of antibodies in clinical trials and/or approved for treatment of solid tumors include: Trastuzumab (antigen: 180 kD, HER2/neu), Edrecolomab (antigen: 40 to 50 kD, Ep-CAM), anti-milk fat globules human (HMFG1) (antigen >200 kD, HMW Mucin), Cetuximab (antigens: 150 kD and 170 kD, EGF receptor), Alemtuzumab (antigen: 21 to 28 kD, CD52), and Rituximab (antigen: 35 kD, CD20 ).
[0017] The antigen targets of Trastuzumab (Her-2 receptor), which is used to treat breast cancer, and Cetuximab (EGF receptor), which is in clinical trials for the treatment of various cancers, are present at some detectable levels in a large number of normal adult human tissues including skin, colon, lung, ovary, liver and pancreas. The safety margin in the use of these therapeutics is possibly provided by the difference in antigen expression levels or in the access of or activity of the antibody at these sites.
[0018] Another type of immunotherapy is active immunotherapy, or vaccination, with an antigen present on a specific cancer(s) or a DNA construct that directs the expression of the antigen, which then evokes the immune response in the individual, that is, it induces the individual to actively produce antibodies against his own cancer. Active immunization has not been used as often as immunotherapy or immunotoxins.
[0019] Several models of disease progression (including cancer) have been suggested. Theories range from cause by a single contagious/transforming event to the evolution of the growing tissue type “like disease” or “like cancer” ultimately leading to one with fully pathogenic or malignant capacity. Some argue that with cancer, for example, a single mutational event is enough to cause malignancy, while others argue that subsequent changes are also necessary. Some others have suggested that increasing mutational burden and tumor grade is necessary for both initiation as well as progression of the neoplasm through a continuum of mutation selection events at the cellular level. Some cancer targets are found only in tumor tissues, while others are present in normal tissues and are regulated and/or overexpressed in tumor tissues. In such situations, some researchers have suggested that overexpression is linked to acquisition of malignancy, while others suggest that overexpression is merely a marker of a trend along a pathway to an increased disease state.
[0020] In some cases, it has been shown that cancer targets, such as oncoproteins expressed or overexpressed in tumors, are present during embryonic and fetal development and serve as a regulator of growth and differentiation. Some researchers have found that the expression of these oncoproteins during embryonic and fetal development appears to be restricted to specific tissues, and also restricted to specific stages of development. In contrast, the expression of these oncoproteins in the adult has been shown to be associated with overexpression in tumor growth and/or a malfunction of tumor suppressor proteins.
[0021] An ideal diagnostic and/or therapeutic antibody would be specific for an antigen present in a large number of cancers but absent or present only at low levels in any normal tissue. The discovery, characterization and isolation of a new antibody capable of binding to an antigen that is specifically associated with cancer(s) would be useful in many ways. First, the antibody would have biological activity against such cancer cells and would be able to recruit the immune system's response to treat the disease in this way. The antibody could be administered as a therapeutic alone or in combination with current treatments, or used to prepare immunoconjugates linked to toxic agents. An antibody with the same specificity but with little or no biological activity when administered alone could also be useful where an antibody could be used to prepare an immunoconjugate with a radioisotope, a toxin or a chemotherapeutic agent or liposome containing a chemotherapeutic agent, with the conjugated form being biologically active by virtue of the antibody targeting the toxin to the antigen-containing cells.
As discussed above, antibodies and other molecules that specifically bind to B7-H3 have been described (see, U.S. Patent No. 7,527,969; 7,368,554; 7,358,354; and 7,279,567; US Patent Applications Nos. US 20090087416; US 20090022747; US20090018315; US2008116219; US20080081346; US 20050202536; US20030103963; US20020168762; PCT Publications Nos. WO 2008/116219; WO 2006/016276; WO 2004/093894; WO 2004/093894; 04/001381; WO 2002/32375; WO 2002/10187 and WO 2001/094413; EP 1292619B; Modak, S. et al (March 1999) "Disialoganglioside GD2 And Antigen 8H9: Potential Targets For Antibody-Based Small Immunotherapy Against Desmoplastic Round Cell Tumor (DSRCT) And Rhabdomyosarcoma (RMS),” Proceedings Of The American Association For Cancer Research Annual Meeting, Vol. 40:474 (90th Annual Meeting Of The American Association For Cancer Research; Philadelphia, Pennsylvania, US; April 10- 14, 1999; Modak, S. et al. (March 2000) “Radioimmunotargeting To Human Rhabdomy osarcoma Using Monoclonal Antibody 8H9,” Proc. Am. Assoc. Cancer Res. 41:724; Modak, S. et al. (2001) “Monoclonal Antibody 8H9 Targets A Novel Cell Surface Antigen Expressed By A Wide Spectrum Of Human Solid Tumors,” Cancer Res. 61(10):4048-4054; Steinberger, P. et al. (2004) “Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains,” J. Immunol. 172(4):2352-2359; Xu, H. et al. (2009) “MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors,” Cancer Res. 69(15):5275-6281).
[0023] Nevertheless, a desirable aspect of an ideal diagnostic and/or therapeutic antibody would be the discovery and characterization of new antibodies capable of mediating and particularly enhancing the activation of the immune system against cancer cells (especially human cancer cells ) that are associated with a variety of cancers. Such compositions would also be useful for drug discovery (e.g., small molecules) and for further characterization of cell regulation, growth, and differentiation.
[0024] Thus, despite all previous advances, a need remains for improved compositions capable of binding to cancer cells and facilitating or mediating an immune response against cancer cells. Such compositions can be used to diagnose and treat such cancers. There is a further need, based on the findings described herein, for new compositions that specifically recognize dual targets on the surface of cells, and which can thereby modulate, either by reducing or increasing, the capabilities of B7-H3 to mediate T cell activation or by recognizing and killing cancer cells expressing B7-H3. An objective of this invention is to identify such compositions. Another goal is to provide new compounds for use in the B7-H3 expression assay.
[0025] As described in detail below, the present invention relates to novel antibodies, including, in particular, dual-affinity targeting reagents ("DARTTMs") comprising B7-H3 T cell activation modulators, which are capable of influence T cell activation as well as new antibodies that bind to the B7-H3 receptors of cancer cells and facilitate or mediate the death of such cells. The present invention is directed to such compositions and their uses in diagnosing and treating diseases such as cancer. SUMMARY OF THE INVENTION:
The present invention relates to antibodies and fragments thereof that are immunoreactive to the mammal, and more particularly to the human B7-H3 receptor and their uses, particularly in the treatment of cancer and inflammation. The invention thus particularly concerns humanized reactive B7-H3 antibodies and their immunoreactive fragments which are capable of mediating and more preferably enhancing the activation of the immune system against cancer cells which are associated with a variety of human cancers.
[0027] In detail, the invention relates to an isolated antibody or immunoreactive fragment thereof, wherein the isolated antibody or fragment comprises a variable domain that specifically binds to an extracellular domain of B7-H3, in which the antibody competes for bind to B7-H3 with any antibodies: BRCA69D, BRCA84D, or PRCA157.
The invention further relates to the isolated antibody described above or immunoreactive fragment thereof, wherein the antibody or fragment comprises a variable domain comprising: (A) CDRi (SEQ ID NO:21), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 25) of the BRCA69D light chain and CDRi (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33) of the BRCA69D heavy chain; (B) CDRi (SEQ ID NO: 5), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9) of the BRCA84D light chain and CDRi (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and BRCA84D heavy chain CDR3 (SEQ ID NO: 17); or (C) CDRi (SEQ ID NO: 37), CDR2 (SEQ ID NO: 39) and CDR3 (SEQ ID NO: 41) of the light chain of PRCA157 and CDRi (SEQ ID NO: 45), CDR2 (SEQ ID NO: :47) and CDR3 (SEQ ID NO:49) of the heavy chain of PRCAi57.
The invention further relates to any of the isolated antibodies described above or immunoreactive fragments thereof, wherein the antibody binds to B7-H3 which is endogenously expressed on the surface of a cancer cell.
The invention further relates to any of the isolated antibodies described above or immunoreactive fragments thereof, wherein the antibody binds to B7-H3 which is internalized by binding to B7-H3 expressed on the surface of the cancer cell.
[0031] The invention further relates to any of the isolated antibodies described above or immunoreactive fragments thereof, which is a humanized monoclonal antibody.
The invention further relates to any of the isolated antibodies described above or immunoreactive fragments thereof, wherein the antibody is a modified antibody comprising a human IgG 1 Fc region, wherein the human IgG 1 variable Fc region comprises at least one amino acid modification with respect to the Fc region of antibody origin, the amino acid modification(s) comprising amino acid modification(s) that alter the affinity or avidity of the variable Fc region to bind to an FcR in a manner that the modified antibody exhibits effector function relative to the natural antibody.
The invention further relates to any of the isolated antibodies described above or immunoreactive fragments thereof, and that the modification of the Fc region comprises: (A) at least one substitution selected from the group consisting of: (1) F243L; (5) Y300L; (2) D270E; (6) V305I; (3) R292P; (7) A330V; and (4) S298N; (8) P396L; (B) at least one substitution of two amino acid residues, the substitutions being selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; and(3) R292P and V305I;(C) at least one substitution of three amino acid residues, the substitutions being selected from the group consisting of: (1) F243L, R292P and Y300L;(2) F243L, R292P and V305I;(3 ) F243L, R292P and P396L; and (4) R292P, V305I and P396L; (D) at least one substitution of four amino acid residues, the substitutions being selected from the group consisting of: (1) F243L, R292P, Y300L and P396L; e(2) F243L, R292P, V305I and P396L; or (E) a substitution of at least five amino acid residues: F243L, R292P, Y300L, V305I and P396.
The invention further relates to the antibody described above, wherein the antibody comprises substitutions of: (A) F243L, R292P, and Y300L; (B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L. The invention further relates to the antibody described above, wherein the antibody comprises: (A) a variable domain comprising CDR1 (SEQ ID NO: 5), CDR2 ( SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9) of BRCA84D light chain and CDRi (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17) of BRCA84D heavy chain ; and(B) a modification of the Fc region comprising the substitutions: L235V, F243L, R292P, Y300L, and P396L.
The invention further relates to the antibody described above, wherein the antibody is a chimeric antibody or a humanized antibody.
The invention further relates to the isolated antibodies described above or immunoreactive fragments thereof, wherein the antibody comprises: (A) a variable light chain having the amino acid sequence of hBRCA84D-2 VL (SEQ ID NO: 89); (B) a variable heavy chain having the amino acid sequence of hBRCA84D-2 VH (SEQ ID NO: 99); and(C) an Fc region that has the substitutions: L235V, F243L, R292P, Y300L, and P396L.
The invention further relates to a hybridoma that secretes a monoclonal antibody that specifically binds to an extracellular domain of B7-H3, in which the antibody competes for the binding of B7-H3 with any of the antibodies: BRCA69D, BRCA84D , or PRCA157.
The invention further relates to a nucleic acid molecule encoding any of the isolated antibodies described above or immunoreactive fragments.
The invention further relates to a dual affinity redirection reagent (DARTTM), the reagent comprising: (A) a polypeptide chain I comprising a specific immunoglobulin VL epitope binding domain for binding to B7-H3 and a specific VH epitope binding domain for binding a molecule other than B7-H3; and(B) a polypeptide II chain comprising an immunoglobulin VH epitope binding domain specific for binding B7-H3 and a VL epitope binding domain specific for binding a molecule other than B7-H3; of polypeptide I and II are associated together so as to form functional epitope binding domains capable of binding to B7-H3 and the non-B7-H3 molecule.
[0040] The invention further relates to the dual affinity redirection reagent (DARTTM) described above, wherein the molecule other than B7-H3 that can be linked to the DARTTM is a hapten, and particularly wherein the hapten is fluorescein isothiocyanate ...
[0041] The invention further relates to the dual affinity redirection reagent (DARTTM) described above, wherein the molecule other than B7-H3 that can be bound by the DARTTM is a T Cell Receptor or the NKG2D receptor.
[0042] The invention further relates to the dual affinity redirection reagent (DARTTM) described above, in which the molecule other than B7-H3 that can be bound to the DARTTM is a tumor associated antigen, and particularly, in which the antigen tumor-associated is selected from the group consisting of A33; ADAM-9; ALCAM; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40/CD154; CD45; CD46; CD5; CD56; CD79a/CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2/GD3/GM2; gp100; HER-2/neu; human papillomavirus-E6; human papillomavirus-E7; Alpha-V-Beta-6 Integrin; JAM-3; KID3; KID31; KSA (17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosaminyltransferase; Oncostatin M; p15; KITE; PSA; PSMA; ROR1; sTn; TNF-β receptor; TNF-α receptor; TNF-Y receptor; Transferrin Receptor; and VEGF receptor.
The invention further relates to a nucleic acid molecule encoding a polypeptide chain of any of the dual affinity redirection reagents (DARTTMs) described above.
The invention further relates to a pharmaceutical composition comprising (i) a therapeutically effective amount of any of the isolated antibodies or immunoreactive fragments described above or dual affinity targeting reagents (DARTTMs) and (ii) a pharmaceutically acceptable carrier.
The invention further relates to the pharmaceutical composition described above, wherein the antibody is a humanized antibody comprising: (A) a variable domain comprising CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9) of the BRCA84D light chain and CDRi (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17) of the BRCA84D heavy chain; and(B) a modification of the Fc region comprising the substitutions: L235V, F243L, R292P, Y300L, and P396L.
The invention further relates to the pharmaceutical composition described above, wherein the antibody is a humanized antibody comprising: (A) a variable light chain having the amino acid sequence of hBRCA84D-2 VL (SEQ ID NO: 89) ;(B) a variable heavy chain having the amino acid sequence of hBRCA84D-2 VH (SEQ ID NO: 99); and (C) an Fc region that has the substitutions: L235V, F243L, R292P, Y300L, and P396L.
[0047] The invention further relates to any of the pharmaceutical compositions described above, which further comprise one or more additional anti-cancer agents, and particularly wherein the additional anti-cancer agent is a chemotherapeutic agent, a radiation therapeutic agent, a therapeutic agent hormone or an immunotherapeutic agent.
[0048] The invention further concerns the use of any of the antibodies or immunoreactive fragments or dual affinity redirection reagents (DARTTMs) described above in the diagnosis of cancer, wherein the isolated antibody, immunoreactive fragment, or DARTTM is detectably labeled.
[0049] The invention further relates to the use described above characterized in that the cancer is characterized by the presence of a cancer cell selected from the group consisting of a cell from a tumor of the glans adrenal, a cancer associated with AIDS, a partial alveolar sarcoma mole, an astrocytic tumor, bladder cancer, bone cancer, brain cancer and spinal cord, a metastatic brain tumor, a breast cancer, carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobic renal cell carcinoma , a clear cell carcinoma, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a small round cell desmoplastic tumor, an ependymoma, an Ewing's tumor, an extraskeletal myxoid chondrosarcoma, an imperfect fibrinogenesis of the bone, a fibrous dysplasia of bone, a cancer of the gallbladder or bile duct, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a cancer. head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's sarcoma, a kidney cancer, a leukemia, a benign lipoma/lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer, a lymphoma , a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasm, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a tumor of parathyroid, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a metastatic renal cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma, a skin cancer, a soft tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a mechanized cancer thyroid gland, and a uterine cancer.
[0050] The invention further concerns the use of any of the antibodies or immunoreactive fragments or dual affinity redirection reagents (DARTTMs) described above in the preparation of a medicament for the treatment or prevention of cancer in a patient. The invention further relates to such uses characterized in that the cancer is characterized by the presence of a cancer cell selected from the group consisting of a cell from a glans adrenal tumor, an AIDS-associated cancer, a soft tissue alveolar sarcoma, a tumor astrocytic, bladder cancer, bone cancer, brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobic renal cell carcinoma, a carcinoma of the stomach clear cell, a colon cancer, a colorectal cancer, a cutaneous benign fibrous histiocytoma, a small round cell desmoplastic tumor, an ependymoma, an Ewing's tumor, an extraskeletal myxoid chondrosarcoma, an imperfect fibrinogenesis of the bone, a fibrous dysplasia of the bone , a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head cancer and neck, hepatocellular carcinoma, an islet cell tumor, a Kaposi's Sarcoma, a kidney cancer, a leukemia, a benign lipoma/lipomatous tumor, a malignant liposarcoma/lipomatous tumor, a liver cancer, a lymphoma, a cancer pulmonary, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasm, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a metastatic renal cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma, a skin cancer, a soft tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a metastatic cancer of the t. thyroid, and a uterine cancer.
[0051] The invention further relates to the uses described above, characterized in that the use further comprises the administration of one or more additional cancer therapies selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, hormonal therapy, and surgery. BRIEF DESCRIPTION OF THE DRAWINGS:
[0052] Figures 1A and 1B show the results of IHC investigations conducted using specimens of normal tissue from the pancreas, liver, lung and colon with BRCA84D at 0.625 μg/ml and 0.078 μg/ml (figure 1A) and normal heart tissue, kidney and adrenal with BRCA84D at 0.625 μg/ml (figure 1B).
Figure 2 shows the results of IHC investigations conducted using pancreatic, breast, cervical, and lung cancer tissue specimens with BRCA84D at 0.625 μg/ml and 0.078 μg/ml.
Figures 3A to 3D show dose-dependent redirected killing mediated by the antibodies of the present invention. Figures 3A and 3B show dose-dependent redirected killing of A498 renal carcinoma cells (with PBMC remaining for 18 hours (LDH)) by monoclonal antibodies reactive against B7-H3 (20:1 effector:target ratio) (Figure 3A : BRCA68D, BRCA69D, PRCA157, GB8, TCR-4420; Figure 3B: OVCA22, BRCA84D, TDH6, TES7, TCR-4420). Figures 3C and 3D show dose-dependent redirected killing of A549 lung cancer cells (with PBMC remaining for 18 hours (LDH)) by monoclonal antibodies reactive against B7-H3 (30:1 effector:target ratio) (Figure 3C: BRCA84D, OVCA22, PRCA157, TES7; Figure 3D: TDH6, BRCA68D, BRCA69D).
[0055] Figures 4A and 4B show the abilities of anti-B7-H3 antibodies to bind to soluble B7H3-2Ig (figure 4A) and soluble B7H3-4Ig B7-H3 (figure 4B) (antibody concentration is 100 nM) . Caption: (A) BLA8; (B) BRCA165; (C) BRCA68D; (D) BRCA69D; (E) BRCA84D; (F) GB8; (G) LUCA1; (H) LUCA50; (I) OVCA21; (J) OVCA22; (K) PA20; (L) PRCA123; (M) SG24; (N) SG27; (O) ST09; (P) TDH4 (184-192); (Q) TDH4; (R) TDH5; (S) TES7. The vertical position of the legend correlates with the position of the corresponding curve.
Figures 5A to 5S demonstrate the binding affinity between the antigens in the solution and the captured monoclonal antibodies (solid lines; B7-H3(4Ig) 100 nM; dashed lines; B7-H3, 100 nM).
Figures 6A to 6I show the results of BIACORE™ analyzes of B7-H3 antibodies immobilized to B7-H3-2Ig (dashed gray lines) or B7-H3-4Ig (solid black lines). Antibodies were titrated from 0.063 µM to 1 µM. Time is in seconds.
Figure 7 provides a comparison of the BIACORETM analysis of PRCA157, BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27 antibodies.
Figure 8 provides an analysis of BIACORETM demonstrating that antibodies BRCA68D, BRCA69D, and PRCA157 do not compete with BRCA84D to bind to human B7-H3.
Figures 9A and 9B show the results of studies on the ability of the anti-B7-H3 antibodies of the present invention to become internalized upon binding to cancer cells (figure 9A, prostate CSC cells; figure 9B, pancreatic Hs700t cells ).
[0061] Figures 10A to 10F show the ability of the anti-B7-H3 antibodies of the present invention to cross one another's block, thus revealing overlapping or distinct epitopes. A ten-fold excess of the competing antibody was employed.
Figures 11A and 11B show the alignment of the amino acid residues of the variable light chains (Figure 11A) or variable heavy chains (Figure 11B) of BRCA84D and its humanized derivative, hBRCA84D.
Figure 12 shows the relative binding affinities of the light chain derivatives hBRCA84D BRCA84D-3VL, BRCA84D-4VL and BRCA84D-5VL to human B7-H3.
Figure 13 shows the relative binding affinities of heavy chain derivatives BRCA84D-2VH, BRCA84D-3VH and BRCA84D-4VH to human B7-H3.
Figure 14 shows the relative binding affinities of (1) antibodies containing hBRCA84D-2VL and hBRCA84D-2VH (assays 1 and 2), (2) chimeric BRCA84D, (3) antibody containing hBRCA84D-5VL and BRCA84D-HC chimeric and (4) antibody containing hBRCA84D-5VL and hBRCA84D-2VH.
Figure 15 shows the ability of Fc-modified humanized anti-B7-H3 antibodies to inhibit HT-1197 urinary bladder carcinoma cell tumor growth in vivo in a murine xenograft model system. The Fc-modified hBRCA84D-2 antibody (comprising Fc modifications L235V, F243L, R292P, Y300L, and P396L) was administered to mice (at a dose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) 7 days, 14 days, 21 days and 28 days after cancer cell implantation.
Figure 16 shows the ability of Fc-modified humanized anti-B7-H3 antibodies to inhibit tumor growth of A498 renal carcinoma cells in vivo in a murine xenograft model system. The Fc-modified hBRCA84D-2 antibody (comprising Fc modifications L235V, F243L, R292P, Y300L, and P396L) was administered to mice (at a dose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) 7 days, 14 days, 21 days and 28 days after cancer cell implantation.
Figures 17A to 17D demonstrate the ability of hBRCA84D-2 / anti-TCR DARTTM ("T-DARTTM") to mediate the redirected killing of SK-MES-1 lung cancer cells, A498 renal carcinoma cells, cancer cells LNCaP prostate and UACC-62 melanoma cells.
Figures 18A to 18C show the pharmacokinetic decay of anti-B7-H3 Mab1 in the serum of tumor-free male mCD16-/-, hCD16A_FOXN1 mice (Figures 18A and 18B). Figure 18C shows the predicted pharmacokinetic profiles generated using a behavioral model 2 with parameters from the 5mg/kg dose at 0.1, 0.5, 1.5, and 10 mg/kg.
[0070] Figure 19 shows the relative expression of HER2 and PRCA135 by the bladder cancer lineage HT-1197.
Figure 20 shows the binding affinity of the hBRCA84D variables of the anti-B7-H3 antibody to HT-1197 cells.
Figures 21A to 21C show the results of a murine xenograft analysis for HT-1197. Groups of 8 female mice received either vehicle or 10 mg/kg IgG control, or centuximab at a dose of 1, 5, or 15 mg/kg or anti-B7-H3 Mab1 antibody at a dose of 0.1, 0.5, 1.5, or 10 mg/kg (Q7D x5). Tumor measurements were taken every 3-4 days. Figure 21A shows the ability of the anti-B7-H3 Mab1 antibody to prevent or inhibit tumor development in the murine xenograft model. Comparisons versus IgG control: Mab1 (1 and 5 mg/kg) versus IgG control *** on day 51; Mab1 (10 mg/kg) versus IgG control ** from day 48. Figure 21B shows the ability of centuximab to prevent or inhibit tumor development in the murine xenograft model. Cetuximab (7 mg/kg) versus IgG Control ** from day 51; Cetuximab (15 mg/kg) versus IgG *** control from day 58. Figure 21C compares the results obtained at the maximum doses tested.
Figures 22A and 22B show the relative expression of HER2 and PMSA by the bladder cancer lineage HT-1376.
Figure 23 shows the results of a murine xenograft analysis for HT-1376. Groups of mice received vehicle or 1.0 mg/kg of anti-B7-H3 Mab1 antibody (Q7D x4).
Figure 24 shows the results of a murine xenograft analysis for AGS. Groups of mice received vehicle or 10 mg/kg of anti-B7-H3 Mab1 antibody at a dose of 0.5, 1.5, or 10 mg/kg (Q7D x5).
Figure 25 shows the results of an in vitro cytotoxicity assay of A549 lung cancer cells incubated with anti-B7-H3 antibodies variable hBRCA84D, chBRCA84D and hBRCA84 (Fc Varl) (Ratio E:T = 25:1; Effector = Human PBMC; LDH Assay reading).
Figure 26 shows the results of a murine xenograft analysis for A549. Groups of mice received vehicle or 1.0 mg/kg of anti-B7-H3 Mab1 antibody (Q7D x4).
Figure 27 shows the results of a murine xenograft analysis for CaLu3. Groups of mice received vehicle or 0.5, 1, or 5 mg/kg (Q7D x5) of anti-B7-H3 Mab1 antibody or IgG control (10 mg/ml).
Figures 28A to 28C show the results of a murine xenograft analysis for LOX-IMVI melanoma cancer cells. Groups of 8 female mice received vehicle or 5 mg/kg of IgG control, or Docetaxel at a dose of 5, 10 or 20 mg/kg or anti-B7-H3 Mab1 antibody at a dose of 0.5, 1.5, or 10 mg/kg. Figure 28A shows the ability of anti-B7-H3 Mab1 antibody to prevent or inhibit tumor development in the murine xenograft model. Figure 28B shows the ability of Docetaxel to prevent or inhibit tumor development in the murine xenograft model. Figure 28C compares the results obtained at the maximum doses tested.
Figure 29 shows the result of a murine xenograft analysis for UACC-62 melanoma cancer cells. Groups of mice received vehicle or 5 mg/kg control IgG, or anti-B7-H3 Mab1 antibody at a dose of 0.5, 1, 5, or 10 mg/kg.
Figures 30A to 30C show the results of a murine xenograft analysis for 2rv prostate cancer cells. Groups of 8 female mice received vehicle or 10 mg/kg of IgG control, or Trastuzumab at a dose of 1.7 or 15 mg/kg or anti-B7-H3 Mab1 antibody at a dose of 0.5, 1.5, or 10 mg/kg. Figure 30A shows the ability of anti-B7-H3 Mab1 antibody to prevent or inhibit tumor development in the murine xenograft model. Figure 30B shows the ability of Trastuzumab to prevent or inhibit tumor development in the murine xenograft model. Figure 30C compares the results obtained at the maximum doses tested.
Figure 31 shows the results of an in vitro cytotoxicity assay of A498 renal cancer cells incubated with anti-B7-H3 antibodies variable hBRCA84D, chBRCA84D and hBRCA84 (Fc Varl) (E:T Ratio = 25:1; Effector = Human PBMC; LDH Assay reading).
Figure 32 shows the result of a murine xenograft analysis for A498 renal cancer cells. Groups of mice received vehicle or 10 mg/kg control IgG, or anti-B7-H3 Mab1 antibody at a dose of 0.1, 0.5, 1.5, or 10 mg/kg. Centuximab (anti-EGRF antibody) was administered to a control group of mice at doses of 1.7, or 15 mg/kg.
Figures 33A and 33B show the result of a murine xenograft analysis for 786-0 renal cancer cells compared to centuximab. Groups of mice received vehicle or 10 mg/kg control IgG, or anti-B7-H3 Mab1 antibody at a dose of 0.1, 0.5, 1.5, or 10 mg/kg. Centuximab (anti-EGRF antibody) was administered to a control group of mice at doses of 1.7, or 15 mg/kg.
Figure 34 shows the result of a murine xenograft analysis for 786-0 renal cancer cells compared to paclitaxel. Groups of mice received vehicle or 5 mg/kg control IgG, or anti-B7-H3 Mab1 antibody at a dose of 0.1, 0.5, 1, 5, or 10 mg/kg. Paclitaxel was administered to a control group of such eight mice at a dose of 2.5 mg/kg. DETAILED DESCRIPTION OF THE INVENTION:
[0086] The present invention relates to antibodies and fragments thereof that are immunoreactive to the mammal, and more particularly to the human B7-H3 receptor and their uses, particularly in the treatment of cancer and inflammation. The invention thus particularly concerns humanized reactive B7-H3 antibodies and immunoreactive fragments thereof which are capable of mediating, and more preferably enhancing, the activation of the immune system against cancer cells which are associated with a variety of human cancers. I. GENERAL TECHNIQUES
[0087] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as, MOLECULAR CLONING: A LABORATORY HANDBOOK, Third Edition (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold Spring Harbor, NY; OLIGONUCLEOTIDE SYNTHESIS: METHODS AND APPLICATIONS (Methods in Molecular Biology), Herdewijn, P., Ed., Human Press, Totowa, NJ; OLIGONUCLEOTIDE SYNTHESIS (Gait, M.J., Ed., 1984); METHODS IN MOLECULAR BIOLOGY, Human Press, Totowa, NJ; CELL BIOLOGY: A LABORATORY BOOKLET (Cellis, J.E., Ed., 1998) Academic Press, New York, NY; ANIMAL CELL CULTURE (Freshney, R.I., Ed., 1987); INTRODUCTION TO CELL AND TISSUE CULTURE (Mather, J.P. And Roberts, P.E., Eds., 1998) Plenum Press, New York, NY; CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Doyle, A. et al, Eds., 1993-8) John Wiley and Sons, Hoboken, NJ; METHODS IN ENZYMOLOGY (Academic Press, Inc.) New York, NY; WEIR'S GUIDE TO EXPERIMENTAL IMMUNOLOGY (Herzenberg, L.A. et al Eds. 1997) Wiley-Blackwell Publishers, New York, NY; MAMMALIAN CELLS GENE TRANSFER VECTORS (Miller, J.M. et al Eds., 1987) Cold Spring Harbor Press, Cold Spring Harbor, NY; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, F.M. et al, Eds., 1987) Greene Pub. Associates, New York, NY; PCR: THE POLYMERASE CHAIN REACTION, (Mullis, K. et al, Eds., 1994) Birkhauser, Boston MA; CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, J.E. et al, eds., 1991) John Wiley and Sons, Hoboken, NJ; SHORT PROTOCOLS IN MOLECULAR BIOLOGY (John Wiley and Sons, 1999) Hoboken, NJ; IMMUNOBIOLOGY 7 (Janeway, C.A. et al 2007) Garland Science, London, UK; Antibodies (P. Finch, 1997) Stride Publications, Devoran, UK; ANTIBODIES: A PRACTICAL APPROACH (D. Catty., ed., 1989) Oxford University Press, USA, New York NY); MONOCLONAL ANTIBODIES: A PRACTICAL APPROACH (Shepherd, P. et al Eds., 2000) Oxford University Press, USA, New York NY; USING ANTIBODIES: A LABORATORY MANUAL (Harlow, E. et al Eds., 1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; ANTIBODIES (Zanetti, M. et al Eds. 1995) Harwood Academic Publishers, London, UK); and DEVITA, HELLMAN, AND ROSENBERG CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY, EIGHTH EDITION, DeVita, V. et al. Eds. 2008, Lippincott Williams & Wilkins, Philadelphia, PA. II. DEFINITIONS
[0088] As used herein, the term "B7-H3" refers to a member of the human B7 protein family, a type I membrane protein with Ig-like domains also known as CD276. The term "2Ig-B7-H3" denotes the form of B7-H3 which only comprises two Ig-like domains; the term "4Ig-B7-H3" denotes the form of B7-H3 which comprises four Ig-like domains (see, Sun, M. et al. (2002) "Characterization of Mouse and Human B7-H3 Genes," J. Immunol 168:6294-6297; Steinberger et al (2004), "Molecular Characterization Of Human 4Ig-B7-H3, A Member Of The B7 Family With Four Ig-Like Domains," J. Immunol. 2004, 172(4) :2352-2359 and Castriconi et al (2004) "Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis;" Proc. Natl. Acad. Sci. (USA ) 101(34): 12640-12645). The “TES7” antigen (WO 2008/066691) is an antigen that shares the characteristics of 4Ig-B7-H3. Consequently, antibodies that specifically bind to TES7 bind to 4Ig-B7-H3. The TES7 antigen can have more than one different epitope, and epitopes can be non-linear. Several anti-B7-H3 antibodies are known to bind to non-linear epitopes, including some only present on the 4Ig-B7-H3 isoform. It is currently believed that TES7 may be overexpressed in certain cancer cells compared to their normal tissue counterparts.
[0089] Agonists, antagonists, and other modulators of B7-H3 function are expressly included within the scope of this invention. Such agonists, antagonists, and modulators are polypeptides that comprise one or more of the antigenic determinant sites of B7-H3, or comprise one or more fragments of such sites, variables of such sites, or peptidomimetics of such sites. Such agonistic, antagonistic and B7-H37 modulatory compounds are provided in linear or cyclic form, and optionally comprise at least one amino acid residue which is not commonly found in nature or at least one amide isostere. These compounds can be glycosylated...
More specifically, the terms "B7-H3 modulators" as used herein are defined as any compound that (1) is capable of interrupting or blocking the interaction between human B7-H3 and its natural ligands or an antibody anti-B7-H3; (2) is capable of binding to human B7-H3 and its natural binders or an anti-B7-H3 antibody; (3) contains an antigenic site that can be used in raising antibodies capable of binding to human B7-H3 and its natural ligands or an anti-B7-H3 antibody; (4) contains an antigenic site that can be used to screen for antibodies capable of binding to human B7-H3 and its natural ligands or an anti-B7-H3 antibody; (5) contains an antigenic site that can be used to raise antibodies capable of interrupting or blocking the interaction between human B7-H3 and its natural ligands or an anti-B7-H3 antibody; (6) contains an antigenic site that can be used to screen for antibodies capable of interrupting or blocking the interaction between human B7-H3 and its natural ligands or an anti-B7-H3 antibody. B7-H3 modulators can be “B7-H3 agonists” or “B7-H3 antagonists” depending on whether their activity increases T cell activation or inhibits T cell activation, respectively.
B7-H3 agonists, antagonists, and modulators include B7-H3 variables, B7-H3 peptide antagonists, peptidomimetics, and small molecules, anti-B7-H3 antibodies and immunoglobulin variables, B7-amino acid variables human H3 including amino acid substitution, deletion and addition variables, or any combination thereof, and chimeric immunoglobulins. The B7-H3 agonists, antagonists and modulators of this invention are based on the identification of B7-H3 domains involved in the binding of human B7-H3 to its natural ligands or anti-B7-H3 antibodies. Thus, the invention provides B7-H3 agonists, antagonists and modulators with molecular structures that duplicate or mimic one or more of the anti-B7-H3 binding domains of human B7-H3.
As used herein, the term "B7-H3 variable" denotes any human B7-H3 amino acid variable, including amino acid substitution, deletion and addition variables, or any combination thereof. The definition encompasses chimeric molecules such as human B7-H3 / non-human chimeras and other hybrid molecules. Also included in the definition is any fragment of a B7-H3 variable molecule that comprises the hybrid variable or region(s) of the molecule.
[0093] As used herein, an "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab", F(ab")2 Fv), single chain (ScFv), mutants thereof, naturally occurring variables, fusion proteins comprising a portion of the antibody with an antigen recognition site of the required specificity, humanized antibodies, chimeric antibodies, "BiTEs®," "DARTTM" molecules and any other modified configuration of the immunoglobulin molecule that comprises a recognition site of antigen of the required specificity.
[0094] The term "BiTEs" (bispecific T cell ligands) refers to a single-stranded molecule having two antigen-binding domains, one of which binds to the T cell antigen and the second binds to one antigen present on the surface of a target (WO 05/061547; Baeuerle, P et al. (2008) "BiTE®: A New Class Of Antibodies That Recruit T Cells" Drugs of the Future 33:137-147; Bargou, et al (2008) “Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody,” Science 321:974-977).
[0095] The term "DARTTM" (Dual Affinity Retargeting Reagent) refers to an immunoglobulin molecule that comprises at least two polypeptide chains that associate (especially through a covalent interaction) to form at least two binding sites of epitopes, which can recognize the same or different epitopes. Each of the polypeptide chains of a DART™ comprises an immunoglobulin light chain variable region and an immunoglobulin heavy chain variable region, but these regions do not interact to form an epitope binding site. Rather, the immunoglobulin heavy chain variable region of one (eg, the first) of the DART™ polypeptide chains interacts with the immunoglobulin light chain variable region of a different (eg, the second) DART™ polypeptide chain to form an epitope binding site. Similarly, the immunoglobulin light chain variable region of one (eg, the first) of the DART™ polypeptide chains interacts with the immunoglobulin heavy chain variable region of a different (eg, the second) DART™ polypeptide chain to form an epitope binding site. DARTTMs can be monospecific, bispecific, trispecific, etc., thus being able to bind simultaneously to one, two, three or more different epitopes (which can be of the same or different antigens). DARTTMs can additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc., thus being able to simultaneously bind one, two, three, four, five, six or more molecules. These two attributes of the DARTTMs (ie degree of specificity and valence can be combined, for example, to produce bispecific antibodies (ie, capable of binding two epitopes) that are tetravalent (ie, capable of binding four sets of epitopes) , etc. DART™ molecules are described in PCT Publications WO 2006/113665, WO 2008/157379, and WO 2010/080538.
[0096] The term "monoclonal antibody" refers to a homogeneous antibody population in which the monoclonal antibody is comprised of amino acids (whether naturally occurring or not) that are involved in selective binding of an antigen. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The term "monoclonal antibody" encompasses not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab", F(ab")2 Fv), single chain (ScFv), their mutants, proteins fusion compounds comprising a portion of the antibody, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity and the ability to bind an antigen. It is not intended to be limited with regard to the source of the antibody or the manner in which it is made (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes total immunoglobulins as well as fragments etc. described above under the definition of “antibody”.
[0097] The term "humanized antibody" refers to a chimeric molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an immunoglobulin from a non-human species and the remaining structure of the molecule's immunoglobulin based on the structure and/or sequence from a human immunoglobulin. The antigen binding site may comprise either complete variable domains fused to constant domains or just complementarity determining regions (CDRs) grafted onto regions of appropriate framework in the variable domains. Antigen binding sites can be wild type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human subjects, but the possibility of an immune response to the external variable region remains (LoBuglio, AF et al. (1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, ” Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach focuses not only on providing human-derived constant regions, but on modifying the variable regions as well as reframing them as closely as possible to the human form. The variable regions of both light and heavy chains are known to contain three complementarity determining regions (CDRs) that vary in response to the antigens in question and determine binding capacity, flanked by four framework regions (FRs) that are relatively conserved in a given species and that putatively provides support for the CDRs. When non-human antibodies are prepared with respect to a particular antigen, the variable regions can be "reshaped" or "humanized" by grafting non-human antibody-derived CDRs into the FRs present in the human antibody to be modified. The application of this approach to various antibodies was reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy" Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity" Science 239:1534-1536; Kettleborough, C.A. et al. (1991) “Humanization Of A Monoclonal Mouse Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation” Protein Engineering 4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma 2:124-134; Gorman, S.D. et al. (1991) “Reshaping A Therapeutic CD4 Antibody,” Proc. Natl. Academic Sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection In Vivo,” Bio/Technology 9:266-271 ; Co, M.S. et al. (1991) “Humanized Antibodies For Antiviral Therapy,” Proc. Natl. Academic Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) "Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy" Proc. Natl. Academic Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J. Immunol. 148: 1149-1154. In some embodiments, humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody that contains all six CDRs of mouse antibodies). In other embodiments, humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered from the original antibody, which is also termed one or more CDRs "derived from" one or more CDRs of the original antibody.
[0098] As used herein, an antibody or polypeptide is said to "specifically" bind a region of another molecule (i.e., an epitope) if it reacts or associates more frequently, more rapidly, with longer duration and/or with greater affinity with that epitope relating to alternative epitopes. For example, an antibody that specifically binds to an epitope of B7-H3 is an antibody that binds to that epitope of B7-H3 with greater affinity, predictably, more readily and/or longer than it binds to other epitopes from B7-H3 or non-B7-H3 epitopes. It is also understood by reading that definition that, for example, an antibody (or portion or epitope) that specifically binds to a first target or cannot specifically or preferentially bind to a second target. As such, "specific binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means “specific” binding.
[0099] As used herein, the term "immunologically active" in reference to an epitope being or "remaining immunologically active" refers to the ability of an antibody (eg, an anti-B7-H3 antibody) to bind to the epitope in different conditions, for example, after the epitope has been subjected to reduction and denaturing conditions.
[00100] Different biological functions are associated with anti-B7-H3 antibodies, including, but not limited to one or more of: an ability to specifically bind to B7-H3 (and in particular B7-H3 molecules that are expressed in surfaces of cancer cells, including, but not limited to, kidney, prostate or lung cancer cells); an ability to competitively inhibit preferential binding of a known anti-B7-H3 antibody to B7-H3, including the ability to preferentially bind to the same B7-H3 epitope to which the original antibody preferentially binds; an ability to bind to a portion of B7-H3 that is exposed on the surface of a living cell in vitro or in vivo; an ability to bind to a portion of B7-H3 that is exposed on the surface of live cancer cells, such as, but not limited to, prostate, lung, or kidney cancer cells; an ability to release a chemotherapeutic agent into cancer cells (such as kidney, prostate, or lung cancer cells) expressing B7-H3 on their surface; and/or an ability to release a therapeutic agent or detectable marker into cancer cells expressing B7-H3 on their surface. As discussed herein, polypeptides (including antibodies) of the invention can have any one or more of these characteristics.
[00101] An "anti-B7-H3 equivalent antibody" or "anti-B7-H3 equivalent polypeptide" refers to an antibody or a polypeptide having one or more biological functions associated with an anti-B7-H3 antibody, such as as, for example, binding specificity.
[00102] As used herein, the term "agent" refers to a biological, pharmaceutical or chemical compound. Non-limiting examples include a complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound. Various compounds can be synthesized, for example, by small molecules and oligomers (eg oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant and animal extracts, and the like.
[00103] The agents that are employed in the methods of this invention may be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen without prior consideration or knowledge of the specific amino acid or other chemical moieties involved in the molecule's association with its natural binding partner(s) or antibodies acquaintances. An example of a randomly selected agent is an agent that is identified through the use and screening of a chemical library or a combinatorial peptide library.
[00104] As used here, an agent is said to be rationally selected or designed when the agent is chosen on a non-random basis that takes into account the sequence of the target location and/or its conformation in connection with the agent's action. With respect to anti-B7-H3 agents, it is currently believed that there are at least three epitopes on B7-H3 against which antibodies can be elevated and therefore at least three sites of action for agents that block the B7 interaction -H3/anti-B7-H3. This invention also encompasses agents that act at the sites of interaction between B7-H3 and its natural binding partner, although other ligands and their active sites interacting with B7-H3 are also encompassed within the scope of this invention, whether currently known or later identified. Agents can be rationally selected or rationally designed by utilizing the peptide sequences that make up the receptor/ligand and/or B7-H3/anti-B7-H3 antibody complex contact sites. For example, a rationally selected peptide agent might be a peptide whose amino acid sequence is identical to an epitope that appears on B7-H3 as it is exposed on the surface of a living cell in its natural environment. Such an agent will reduce or block the association of the anti-B7-H3 antibody with B7-H3, or the association of B7-H3 with its natural ligand, as desired, through binding to the anti-B7-H3 antibody or natural ligand.
[00105] As used herein, the term "labelled," with respect to an antibody, is intended to encompass direct labeling of the antibody through coupling (i.e., physically binding) a detectable substance, such as a radioactive agent or a fluorophore (e.g. , phycoerythrin (PE) or fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC)) to the antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance.
[00106] As used herein, the term "association", with respect to an antibody, includes covalent and non-covalent attachment or attachment of an agent (e.g., chemotherapeutic agent) to the antibody. The antibody can be associated with an agent (eg, chemotherapeutic agent) by direct binding or indirect binding through the attachment to a common platform, such that the antibody directs the location of the agent to the cancer cell to which the antibody binds and into that the antibody and agent do not substantially dissociate under psychological conditions so that the agent is not targeted to the same cancer cell to which the antibody binds or so that the potency of the agent is not reduced.
[00107] The term "biological sample" encompasses a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay. The definition encompasses saliva, blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived from them, and their progeny, for example, cells obtained from a tissue sample collected from an individual suspected of having cancer, in preferred embodiments of ovarian, lung, prostate, pancreas, cervix, and breast tissue. The definition also includes samples that have been manipulated in any way after obtaining them, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins or polynucleotides, or incorporating into a solid or semi-solid matrix for sectioning purposes. The term "biological sample" encompasses a clinical sample, and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluid, and tissue samples.
[00108] The term "host cell" includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with polynucleotide(s) of this invention.
[00109] As used herein, the term "delayed metastasis development" means to delay, prevent, reduce, delay, stabilize and/or delay metastasis development. This delay can be for different periods of time, depending on the cancer history and/or the individual being treated. As is evident to one skilled in the art, sufficient or significant delay may actually encompass prevention, in which the individual does not develop metastasis.
[00110] As used herein, an "effective amount" of a pharmaceutical composition, in one embodiment, is an amount sufficient to effect beneficial or desired results including, without limitation, clinical results such as shrinkage in tumor size (in the context of cancer , for example, breast or prostate cancer), retarded cancer cell growth, delayed metastasis development, decreased symptoms resulting from the disease, increased quality of life for those suffering from the disease, decreased dose of other medications required for treating the disease, enhancing the effect of other medication such as through targeting and/or internalizing, delaying the progress of the disease, and/or prolonging the individuals' survival. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound or pharmaceutical composition is an amount sufficient to reduce the proliferation (or destruction) of cancer cells and to reduce and/or delay the development, or growth, of cancer cell metastases, either directly or indirectly. In some embodiments, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more chemotherapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result is can be or is achieved. While individual needs vary, determining optimal ranges of effective amounts of each component is within the skill of the subject. Typical dosages range from 1 to 100 mg/kg/body weight. The most preferred dosages comprise from 10 to 100 mg/kg/body weight.
As used herein, an agent or nucleic acid molecule, antibody, composition or cell, etc., is said to be "isolated" when that nucleic acid molecule, agent, antibody, composition, or cell, etc. is substantially separated from contaminating nucleic acid molecules, antibodies, agents, compositions, or cells, etc. naturally present in its original source.
[00112] The term "individual" refers to a vertebrate animal, preferably a mammal. Mammals include, but are not limited to, humans, farm animals, sport animals, pets, primates, mice and rats. In the most preferred embodiment, the term individual means a human.
[00113] The terms "polypeptide," "oligopeptide," "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based on an antibody, the polypeptides can occur as single chains or as associated chains.
Also encompassed within the scope of the invention are the peptidomimetics of the B7-H3 peptide agonists, antagonists and modulators (including anti-B7-H3 antibodies) described herein. Such peptidomimetics include peptides in which at least one amino acid residue is replaced with an amino acid residue that is not commonly found in nature, such as the D-isomer of the amino acid or an N-alkylated species of the amino acid. In other embodiments, peptidomimetics are constructed by replacing at least one amide bond (-C(=O)-NH-) in a B7-H3 peptide agonist, antagonist or modulators with an amide isostere. Suitable starch isosteres include -CH2-NH-, -CH2-S-, -CH2-S(O)-, -CH2-S(O)2-, -CH2-CH2- -CH=CH- (E form or Z), -C(=O)-CH2- -CH(CN)-NH--C(OH)-CH2-, and -OC(=O)-NH-. Amide bonds in a B7-H3 agonist, antagonist or peptide modulator that are suitable candidates for substitution with amide isosteres include bonds that are hydrolysable by the endogenous esterases or proteases of the intended subject of the agonist, antagonist or peptide modulator treatment B7-H3.
[00115] As used herein, the term "substantially pure" refers to material that is at least 50% pure (ie, free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, more preferably at least 99% pure, and most preferably more than 99% pure.
[00116] As used herein, the term "toxin" refers to any substance that effects an adverse response within a cell. For example, a toxin targeted at a cancer cell would have an adverse, sometimes detrimental, effect on the cancer cell. Examples of toxins include, but are not limited to, a taxane, a maytansinoid, an auristatin (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (such as those described in U.S. Patent Nos. 5,208,020; 5,416,064; 6,333,410; 6,340,701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497; 7,585,857; or 7,851,432), a calicheamicin, an anthracycline (for example, doxorubicin), a CC-1065 analogue, docetaxel; cathepsin B or E; ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, and RNase; radiolabeled antibodies (eg, tiuxetan conjugated or labeled with a toxic radioisotope (eg, 90Y; 131I, 177Lu, 186Re, 188Re, 211At, 212Bi, 213Bi, 225Ac, etc.).
[00117] As used herein, the terms "treatment" or "treating" means an approach to achieving a beneficial or desired outcome including, and preferably, a beneficial or desired clinical outcome. Such beneficial or desired clinical outcomes, but are not limited to, one or more of the following: reduced proliferation of (or destruction) cancer cells or other diseases, reduced metastasis of cancer cells found in cancers, shrinkage of tumor size, decrease of symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging the survival of individuals.
[00118] As used herein, the term cancer is intended to encompass cancers characterized by the presence of a cancer cell selected from the group consisting of a glans adrenal tumor cell, an AIDS-associated cancer, a soft tissue alveolar sarcoma, an astrocytic tumor , bladder cancer (squamous cell carcinoma and transitional cell carcinoma), bone cancer (adamantinoma, aneurysmal bone cysts, osteochondroma, osteosarcoma), brain and spinal cord cancer, a metastatic brain tumor, a breast cancer, carotid body tumors , a cervical cancer, a chondrosarcoma, a chordoma, a chromophobic renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a benign cutaneous fibrous histiocytoma, a small round cell desmoplastic tumor, an ependymoma, an Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a fibrinogenesis imperfecta of the bone, a fibrous dysplasia of the bone, a cancer of the vein. gallbladder or bile duct, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head and neck cancer, hepatocellular carcinoma, an islet cell tumor, a Kaposi's sarcoma, a kidney cancer (nephroblastoma, papillary renal cell carcinoma), a leukemia, a benign lipoma/lipomatous tumor, a liposarcoma/malignant lipomatous tumor, a liver cancer (hepatoblastoma, hepatocellular carcinoma), a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasm, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary thyroid carcinoma, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor , a phaeochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a rare hematologic disorder, a metastatic kidney cancer, a rhabdoid tumor, a ra. bdomyosarcoma, a sarcoma, a skin cancer, a soft tissue sarcoma, a squamous cell cancer, a stomach cancer, a synovial sarcoma, a testicular cancer, a thymic carcinoma, a thymoma, a metastatic thyroid cancer, and a cancer uterine (cervix carcinoma, endometrial carcinoma, and leiomyoma). III. METHODS OF MANUFACTURING ANTIBODIES AND POLYPEPTIDES
[00119] Methods of manufacturing monoclonal antibodies are known in the art. One method that can be employed is the Kohler, G. et al. (1975) "Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity" Nature 256:495-497 or its modification. Typically, monoclonal antibodies are developed in non-human species, such as mice. Usually a mouse or rat is used for immunization, but other animals can also be used. Antibodies are produced by immunizing mice with an immunogenic amount of cells, cell extracts, or protein preparations that contain human B7-H3. The immunogen can be, but is not limited to, primary cells, cultured cell lines, cancer cells, nucleic acids, or tissues. In one embodiment, human lung carcinoma cells are used. Cells used for immunization, eg human testes or pancreatic adenocarcinoma or stomach cells, can be cultured for a period of time (eg at least 24 hours) before their use as an immunogen. The cells (for example, cells from human testis, stomach, or pancreatic adenocarcinoma) can be used as immunogens by themselves or in combination with a non-denaturing adjuvant such as Ribi. In general, cells must be kept intact and preferably viable when used as immunogens. Intact cells may allow antigens to be better detected than cells disrupted by the immunized animal. The use of denaturing or harsh adjuvants, eg Freud's adjuvant, can break cells and therefore be discouraged. The immunogen can be administered several times at periodic intervals such as fortnightly or weekly, or it can be administered in such a way to maintain viability in the animal (eg, in a tissue recombinant).
[00120] In one embodiment, monoclonal antibodies that bind to B7-H3 are obtained using host cells that overexpress B7-H3 as an immunogen. Such cells include, by way of example and not limitation, human lung carcinoma cells and human colon cancer cells.
[00121] To monitor the antibody response, a small biological sample (eg blood) can be obtained from the animal and tested for antibody titration against the immunogen. The spleen and/or several large lymph nodes can be removed and dissociated into single cells. If desired, spleen cells can be selected (after removal of non-specifically adherent cells) by applying a cell suspension to a plate or a well coated with the antigen. B cells, which express antigen-specific membrane-bound immunoglobulin, will stick to the plate, and are not washed with the rest of the suspension. The resulting B cells, or all dissociated spleen cells, can then be fused with myeloma cells (eg, X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, CA). Polyethylene glycol (PEG) can be used to join spleen and lymphocytes with myeloma cells to form a hybridoma. The hybridoma is then cultured in a selective medium (eg, hypoxanthine, aminopterin, thymidine medium, also known as “HAT medium”). The resulting hybridomas are then placed on plates limiting the dilution, and are assayed for the production of antibodies that specifically bind to the immunogen, using, for example, FACS (fluorescence activated cell separation) or immunohistochemistry (IHC) screening . Hybridomas that secrete selected monoclonal antibody are then cultured either in vitro (eg, in tissue culture bottles or hollow fiber reactors), or in vivo (eg, as ascites in mice).
[00122] As another alternative to the cell fusion technique, B cells immortalized by the Epstein-Barr Virus (EBV) can be used to produce monoclonal antibodies of the present invention. Hybridomas are expanded and subcloned, if desired, and supernatants are assayed for anti-immunogenic activity by standard assay procedures (eg, FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, etc.).
[00123] In another alternative, anti-B7-H3 monoclonal antibody and any other equivalent antibodies can be sequenced and produced recombinantly by any means known in the field (eg, humanization, use of transgenic mice to produce fully human antibodies, technology of phage display, etc.). In one embodiment, the anti-B7-H3 monoclonal antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use.
[00124] The polynucleotide sequence of anti-B7-H3 monoclonal antibody and any other equivalent antibodies can be used for genetic manipulation to generate a "humanized" antibody to improve the affinity or other characteristics of the antibody. The general principle in humanizing an antibody involves retaining the basic sequence of the antigen-binding portion of the antibody, while exchanging the non-human remainder of the antibody with human antibody sequences. There are four general steps to humanizing a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequence of the light and heavy variable domains of the starting antibody (2) designing the humanized antibody, that is, deciding which region of the antibody framework to use during the humanization process (3 ) humanize current methodologies/techniques and (4) transfect and express the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.
[00125] A number of "humanized" antibody molecules comprising an antigen binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies that have rodent or rodent modified V regions and their associated complementarity determining regions (CDRs ) joined to the human constant domains (see, for example, Winter et al. (1991) "Man-made Antibody," Nature 349:293-299; Lobuglio et al. (1989) "Mouse/Human Chimeric Antibot monoclonal In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989), Shaw et al. (1987) "Characterization Of A Mouse/Human Chimeric Antibody Monoclonal (17-1 A) To A Colon Cancer Tumor-Associated Antigen,” J. Immunol. 138:4534-4538, and Brown et al. (1987) “Tumor-Specific Genetically Engineered Murine/Human Chimeric Antibody monoclonal,” Cancer Res. 47:3577-3583) . Other references describe rodent CDRs grafted onto a human scaffolding region (FR) prior to fusion with an appropriate human antibody constant domain (see, for example, Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,” Science 239:1534-1536; and Jones et al. (1986) “Replacing The Complementarity -Determining Regions In A Human Antibody With From A Mouse,” Nature 321:522-525). Another reference describes rodent CDRs supported by recombinantly clad rodent framework regions. See, for example, European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize the unwanted immune response to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of those moieties in human recipients. Other methods of humanizing antibodies that can also be used are described by Daugherty et al. (1991) “Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antiboy Directed Against The CD18 Component Of Leukocyte Integrins,” Nucl. Acids Res. 19:2471-2476 and in U.S. Patents 6,180,377;6,054,297; 5,997,867; and 5,866,692.
The invention also encompasses single chain variable region ("scFv") fragments of antibodies of this invention, such as mu-anti-B7-H3. Single chain variable region fragments are made by linking light and/or heavy chain variable regions using a short linker peptide. Bird et al. (1988) ("Single-Chain Antigen-Binding Proteins," Science 242:423426) describe exemplary binding peptides that cleave approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of another variable region. Binders from other sequences have been designed and used (Bird et al. (1988) "Single-Chain Antigen-Binding Proteins," Science 242:423-426). Binders can, in turn, be modified for additional functions such as drug fixation or fixation to solid supports. Single-chain variables can be produced either recombinantly or synthetically. For scFv synthetic production, an automatic synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide encoding the scFv can be introduced into a suitable host cell, eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. Polynucleotides encoding the scFv of interest can be made through routine manipulations such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification technique known in the art.
[00127] The invention includes modifications to antibodies and polypeptides that bind to B7-H3 and its agonists, antagonists, and modulators, including functionally equivalent antibodies and polypeptides that do not significantly affect their properties and variables that had increased or decreased activity. Modification of polypeptides is routine practice in the art and need not be described in detail here. Examples of modified polypeptides include polypeptides with conservative amino acid residue substitutions, one or more deletions or additions of amino acids that do not significantly detrimentally change functional activity, or use of chemical analogs. Amino acid residues that can be conservatively substituted for one another include, but are not limited to: glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine; lysine/arginine; and phenylalanine/triosine. Such polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Preferably, the amino acid substitutions would be conservative, that is, the substituted amino acid would have similar chemical properties as that of the original amino acid. Such conservative substitutions are known in the art, and examples have been provided above. Amino acid modification can range from altering or modifying one or more amino acids to complete the redesign of a region, such as the variable region. Variable region changes can alter binding affinity and/or specificity. Other methods of modification include using coupling techniques known in the art, including, but not limited to, enzymatic means, substitution and oxidative chelation. Modifications can be used, for example, to attach labels for immunoassay, such as attaching radioactive moieties for radioimmunoassay. Modified polypeptides are made using established procedures in the art and can be selected using standard assays known in the art.
The invention also encompasses fusion proteins comprising one or more fragments or regions of the polypeptides and antibodies of this invention. In one embodiment, a fusion polypeptide is predicted to comprise at least 10 contiguous light chain variable region amino acids and at least 10 heavy chain variable region amino acids. In another embodiment, a fusion polypeptide contains a heterologous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains a light chain variable region and a heavy chain variable region from an antibody produced from a publicly deposited hybridoma. For purposes of this invention, an antibody fusion protein contains one or more polypeptide domains that specifically bind to B7-H3 and another amino acid sequence which is not attached in the natural molecule, for example, a heterologous sequence or a homologous sequence from another region.
[00129] An anti-B7-H3 polypeptide, and other agonists, antagonists and modulators of B7-H3 can be created by methods known in the art, for example, synthetically or recombinantly. One method of producing B7-H3 peptide agonists, antagonists, and modulators involves chemical synthesis of the polypeptide, followed by treatment under appropriate oxidizing conditions to obtain the natural conformation, ie, the correct disulfide bonds. This can be accomplished using methodologies well known to those skilled in the art (see, for example, Kelley, RF et al. (1990) in: METHODS AND PRINCIPLES OF GENETIC ENGINEERING, Setlow, JK Ed., Plenum Press, NY, vol. 12 , pp 1-19; Stewart, JM et al (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL; see also U.S. Patent Nos. 4,105,603; 3,972,859; 3,842. 067; and 3,862,925).
[00130] Polypeptides of the invention can be conveniently prepared using solid phase peptide synthesis (Merrifield, B. (1986) "Solid Phase Synthesis," Science 232(4748):341-347; Houghten, RA (1985) " General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids," Proc. Natl. Acad. Sci. (USA) 82(15):5131-5135; Ganesan, A. (2006) "Solid-Phase Synthesis In The Twenty-First Century" Mini Rev. Med. Chem. 6(1):3-10).
[00131] In yet another alternative, fully human antibodies can be obtained through the use of commercially available mice that have been designed to express specific human immunoglobulin proteins. Transgenic animals that are designed to produce a more desirable (eg, fully human) or more robust immune response can also be used to generate human or humanized antibodies. Examples of such technology are XENOMOUSETM (Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE® and TC MOUSETM (both from Medarex, Inc., Princeton, NJ).
[00132] In an alternative, the antibodies can be made recombinantly and expressed using any method known in the art. Antibodies can be made recombinantly by first isolating the antibodies made from host animals, obtaining the gene sequence and using the gene sequence to express the antibody recombinantly in host cells (eg, CHO cells). Another method that can be employed is to express the antibody sequence in plants (eg, tobacco) or transgenic milk. Suitable methods for expressing antibodies recombinantly in plants or milk have been described (see, for example, Peeters et al. (2001) "Production Of Antibodies And Antibody Fragments In Plants," Vaccine 19:2756; Lonberg, N. et al. al (1995) "Human Antibodies From Transgenic Mice," Int. Rev. Immunol 13:65-93; and Pollock et al (1999) "Transgenic Milk As A Method For The Production Of Recombinant Antibodies," J. Immunol Methods 231:147-157). Suitable methods for making antibody derivatives, e.g., humanized, single chain, etc. are known in the art. In another alternative, the antibodies can be made recombinantly by phage display technology (see, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; 6,265,150; and Winter, G. et al (1994) "Making Antibodies By Phage Display Technology" Annu. Rev. Immunol. 12.433-455).
[00133] The antibodies or protein of interest can be subjected to sequencing by Edman degradation, which is well known to those skilled in the art. Peptide information generated from Edman mass spectrometry or degradation can be used to design probes and primers that are used to clone the protein of interest.
[00134] An alternative method of cloning the protein of interest is by "filtering" using purified B7-H3 or portions thereof to cells expressing the antibody or protein of interest. B7-H3 exists in a “2Ig” form and as a “4Ig” form. The amino acid sequence of the "2Ig" form of human B7-H3 is (SEQ ID NO:1): ML .RR RG SF GM GVfl VG A AT ,GAI ,W FC LT GA I. E VQVFE D PWA T ,VG TD AT LCC SFSPEPGFSL AQLNLIWQLT DTKQLVH5FA EGQDQGSAYA NRTALFPDLL AQGNASLRW RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG LFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQ PMTFPPEALW VTVGLSVCLI ALLVALAFVC WRKIKQSCEE ENAGAEDQDG EGEGSKTALQ PLKHSDSKED DGQEIA
[00135] The cDNA sequence encoding the Form "21g" "of B7-H3 is human (SEQ ID N0: 2): atgctgcgtc ggcggggcp.g ccctggcatg ggtgtgcatg tgggtgαagc cctgggagca ctgtggttct gcctcacagg agccctggag gtααaggtcc ctgaagaccc agtggtggca ctggtgggca ccgatgccac cctgtgctgc tccttctccc ctgagcctgg cttcagcctg gcacagctca acctcatctg gcagctgaca gataccaaac agctggtgca cagctttgct gagggccagg accagggcag cgcctatgcc aaccgcacgg ccctcttccc ggacctgctg gcacagggca acgcatccct gaggctgcag cgcgtgcgtg tggcggacga gggcagcttc acctgcttcg tgagcatccg ggatttcggc agcgctgccg tcagcctgca ggtggccgct ccctactcga agcccagcat gaccctggag cccaacaagg acctgcggcc aggggacacg gtgaccatca cgtgctccag ctaccggggc taccctgagg ctgaggtgtt ctggcaggat gggcagggtg tgcccctgac tggcaacgtg accacgtcgc agatggccaa cgagcagggc ttgtttgatg tgcacagcgt cctgcgggtg gtgctgggtg cgaatggcac ctacagctgc cbggLgcgtd accccgtgct gcagcaggat gcgcacggct ctgtcaccat αaαagggcag cctatgacat tccccccaga ggccctgtgg gtgaccgtgg ggctgtctgt ctgtctcatt gcactgctgg tggccctggc tttcgtgtgt tggagaaaga tcaaaca gag ctgtgaggag gagaatgcag gagctgagga ccaggatggg gagggagaaq gctccaagac agccctgcag cctctgaaac actctgacag caaagaagat gatggacaag aaatagcc
The amino acid sequence of the "2Ig" form of human B7-H3 (SEQ ID NO:1) (shown in bold and underlined below) is fully included in the "4Ig" form of human B7-H3 (SEQ ID NO: 76): MLRRRGSPGM GVHVGAALGA LWFCLTGAL- VQVPEDPWA LVGTDATLCC SFSPEPGFSL AQLNLIWQLT DTKQLVH5FA EGQDQGSAYA NRTALFPDLL AQGTJASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE PNKDLRPGDT VTITCSSYRG YPEAEVFWOD GQGVPLTGNV TTSQMANEQG LF ■ DVH S1LRV V LGANGTYSC LVRN VLQQD AH ££ VTIT PQ RSPIGAVEVQ VPEDPWALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG RDQGSAYANR TALFPDLLAQ GtJASLRLQRV RVADEG3FTÇ FVSIRDFGSA ÃVSLQVAAPY SKPSMTLBPN KDLRPGDTVT ITCSSYRGYP RAEVFWQDGQ GVPLTGNVTT SQMANEQGLF Ç>VH3VLRWIJ GANGTYSCLV RNFVIQQDAH GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSDGETALQGEN QEGSA
[00137] The cDNA sequence encoding the "4Ig" form of human B7-H3 is (SEQ ID NO:77); the residues encoding the shape "2Ig" B7-H3 are shown in bold and underlined: atgctgcgte ggcggggcag ccctggoatg ggtgtgcatq tgggtqcaqc cctggqaqca ctqtqqttct gcctcacagg aqccctgqa ggt cca ggt cc ctgaaga ccc agtggtggca ctggtgggca ccgatgccac cctgtgctgc tccttctccc ctgagcctgg cttcagcctg gcacagctca acctcatctg gcagctgaca gataccaaac agctggtgca cagctttgct gagggccagg accagggcag cgcctatgcc aaccgcacgg αcctottccc ggaαctgctg gcacagggca acgcatccct gaggctgcag cgcgtgcgtg tggcggacga gggcagcttc acctgcttcg tgagcatccg ggatttcggc agcgctgccg tcagcctgca ggtggccgct ccctactcga agcccagcat gaccctggag eecaaeaagg acctgcggcc aggggacacg gtgaccatca cgtgctccag ctaccagggc taccctgagg ctgaggtgtt ctggcaggat gggcagggtg tgcccctgac tggcaacgtg accacgtcgc agatggccaa cgagcagggc ttgtttgatg tgcacagcat cctgcgggtg gtgctgggtg CAAA-ggcac ctacagctgc ctggtgcgca accccgtgct gcagcaggat gc gc Eager, ctgtcaccat cacaccccag agaagcccca caggagccgt ggaggtccag gtccctgagg acccggtggt ggccctagtg ggcaccgatg ccaccctqcq ctqctccttc tcc saαgaga ctqqcttcaq αciLqqcacaq ctπaacctca tctqqcaqct qacagacacc aaasaqatqg tqcacagttt caccqaagqc cgggaccagg gcagcgccta tgccaaaαgci aαggcsaatat taccggaaat gatggaacaa ggeaatgeat ccctαagqct qeagcqoqtq cqtqtqqcqg acgaqggcaq cttcacctqc ttcqtgaqca tccgqqattt ogqcaqogct gccgtcagcc tgcaggtggc cgctccctac tcgaagccca gcatgaccct gqagcccaac aaggacctgc ggccagggga cacggtgacc atcacqtgct ccagctaccg gggctaccct gaggetgagg tgttctggca ggatgggcag g ~ gtg ^ gcccc tgactggcaa cgtgaccacg tcgcagatgg ccaacgagca gggcttgttt gatgtgcaca gcgtcctgcg ggtggtgctg ggtgcgaatg gcacctacag ctgcctggtg □ qcaacππcq tcctqcagca ggatgcgcac ggctctgtca ccatcacagg gcagcctatg a.cattccccc ca.qa.gqccct qtqqqtgacscs gtgqqgatgt argrergtat aattgaactg ctggtggccc tggctttcgt gtgctggaga aagatcaaac agagctgtga ggaggagaat gcaggaqctq AGGA-ccaqga tggggaggga gaaggctcca agacagccct gcagcctctg to-AACA-ctctg acagcaaaga agatgatgga cc caagaaatag
[00138] The "Filtering" procedure can be conducted by obtaining a cDNA library from tissues or cells expressing B7-H3, overexpressing the cDNAs in a second cell type, and screening the transfected cells from the second cell type for a specific binding to B7-H3. Detailed descriptions of methods used in cloning mammalian genes encoding cell surface proteins by "filtering" can be found in the art (see, for example, Aruffo, A. et al. (1987) "Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System," Proc. Natl. Acad. Sci. (USA) 84:8573-8577 and Stephan, J. et al. (1999) "Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation,” Endocrinol. 140:5841-5854).
cDNAs encoding anti-B7-H3 antibodies, and other B7-H3 peptide agonists, antagonists and modulators can be obtained by reverse transcription of the mRNAs of a particular cell type according to standard methods in the art. Specifically, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures set forth in Sambrook et al. supra or extracted by commercially available nucleic acid binding resins following the accompanying instructions provided by the manufacturers (e.g., Qiagen, Invitrogen, Promega). The synthesized cDNAs are then introduced into an expression vector to produce the antibody or protein of interest in cells of a second type. It is implied that an expression vector must be replicable in host cells either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, and cosmids.
[00140] Vectors containing the polynucleotides of interest can be introduced into the host cell by any number of appropriate means, including electroporation, transfection, employment of calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (for example, where the vector is an infectious agent such as vaccinia virus). The choice of introducer vectors or polynucleotides will often depend on aspects of the host cell.
[00141] Any host cells capable of overexpressing the heterologous DNAs can be used in order to isolate the genes encoding the antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. Preferably, host cells express the cDNAs at a level about 5 times higher, more preferably 10 times higher, even more preferably 20 times higher than that of the corresponding endogenous antibody or protein of interest, if present, in the cells hostesses. Screening host cells for specific binding to B7-H3 is performed by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.
[00142] Various techniques are also available that can now be employed to produce agonists, antagonists, and modulators of mutant B7-H3 peptide that encode additions, deletions or changes in the resulting protein amino acid sequence relative to the agonist, antagonist or molecule. principal B7-H3 peptide modulator.
The invention includes polypeptides comprising an amino acid sequence of the antibodies of this invention. The polypeptides of this invention can be made by procedures known in the art. Polypeptides can be produced by proteolytic or other degradation of antibodies, by recombinant methods (i.e., single or fusion polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an anti-B7-H3 polypeptide could be produced by an automated polypeptide synthesizer employing the solid phase method. IV. METHODS FOR SCREENING POLYPEPTIDES AND MONOCLONAL ANTIBODIES
[00144] Various methods can be used to screen for polypeptides and monoclonal antibodies that bind to B7-H3. It is understood that "binding" refers to specific biologically or immunologically relevant binding, and does not refer to non-specific binding that can occur, for example, when an immunoglobulin is used in a very high concentration against a non-specific target. In one embodiment, monoclonal antibodies are screened for binding to B7-H3 using standard screening technique. In this way, the anti-B7-H3 monoclonal antibody was obtained. Preferred hybridomas of the present invention are those that produce BRCA69D, BRCA84D or PRCA157 antibodies.
[00145] Additional monoclonal antibodies that bind to B7-H3 can be identified. To this end, monoclonal antibodies are screened for their differential ability to bind to cancerous tissues but not to non-cancer cells. In one embodiment, monoclonal antibodies that bind to B7-H3 and that also cross-react with human cancer cells or tissues, but not normal cells or tissues to the same degree, are selected. One method that can be used for screening is immunohistochemistry (IHC). Standard immunohistochemistry techniques are known to those of ordinary skill in the art. See, for example, ANIMAL CELL CULTURE METHODS (J.P. Mather and D. Barnes, eds., Academic Press, NY, Vol. 57, Ch. 18 and 19, pp. 314-350, 1998). Biological samples (eg tissue) can be obtained from biopsies, autopsies, or necropsy. To check whether B7-H3 is present only in cancer cells, anti-B7-H3 antibodies can be used to detect the presence of B7-H3 in the tissues of individuals with cancer, while other non-cancerous tissues of the individual suffering from cancer or tissues from individuals without cancer are used as a control. The fabric can be integrated into a solid or semi-solid substance that prevents damage during freezing (eg, agarose gel or OCT) and then sectioned for dyeing. Cancers from different organs and to different degrees can be used to screen for monoclonal antibodies. Examples of tissues that can be used for screening purposes include, but are not limited to, ovary, breast, lung, prostate, cervix, kidney, skin, thyroid, brain, heart, liver, stomach, nerve, blood vessels, bone, upper digestive tract and pancreas. Examples of different types of cancer that can be used for screening purposes include, but are not limited to, carcinomas, adenocarcinomas, sarcomas, adenosarcomas, lymphomas, and leukemias.
[00146] In yet another alternative, cancer cell lines such as HMEC (BioWhittaker CC-2251), HUVEC (primary edothelial cells), BT-474 (ATCC# HTB-20), MCF7 (ATCC# HTB22), MDA- MB-175-VII (ATCC# HB-25), MDA-MB-361 (ATCC# HB-27), SKBR3 (ATCC# HTB-30), A549 (ATCC# CCL-185), Calu-3 (ATCC# HTB-55), SKMES-I (ATCC# HTB-58), ES-2 (ATCC# CRL-1978), SKOV3 (ATCC# HTB-77), Panc-1 (ATCC# CRL-1469), AsPC-I (ATCC# CRL-1682), HPAF-II (ATCC# CRL- 1997), Hs700T (ATCC# HTB-174), Colo205 (ATCC# CCL-222), HT-29 (ATCC# HTB-38), SW480 ( ATCC# CCL-228), SW948 (ATCC# CCL-237), 293 (ATCC# CRL-1573), 786-0 (ATCC# CRL-1932), A498 (ATCC# HTB-44), Caki-2 (ATCC # HTB-47), COS-7 (ATCC # CRL-1651), RL-65 (ATCC # CRL-10345), SV-T2 (ATCC # CCL-163.1), 22RV1 (ATCC # CRL-2505), DU145 (ATCC# HTB-81), LNCaP (ATCC# CRL-1740), PC-3 (ATCC# CRL-1435), HT29 (ATCC# HTB-38), Hs746T (ATCC# HTB-135), NCI-N87 (ATCC# CRL-5822) and normal cells from their respective tissues can be used to screen for antibodies. monoclonal ones that are specific for cancer tissue. First, or low passage, cell cultures derived from normal tissues from different organs including, but not limited to, kidney, ovary, breast, lung, prostate, colon, kidney, skin, thyroid, aortic smooth muscle, and edothelial cells can be used as negative controls. Cancerous or non-cancerous cells can be grown on glass slides or coverslips, or on plastic surfaces, or prepared on a CellArrayTM device, as described in WO 01/43869, and screened for antibody binding using IHC as described above for the fabrics. Alternatively, cells can be removed from the growth surface using non-proteolytic media and spun into a pellet, which is then integrated and treated as tissues for IHC analysis as described above. Cells can be inoculated into immunodeficient animals, a tumor allowed to grow, and then that tumor can be harvested, integrated and used as a tissue source for IHC analysis. In another alternative, single cells can be screened by incubation with the primary antibody, a secondary "reporter" antibody linked to a fluorescent molecule and then analyzed using a fluorescent activated cell sorting machine (FACS).
[00147] Any of several detection systems can be used to detect the binding of antibodies to tissue section. Typically, immunohistochemistry involves binding a primary antibody to tissue and then a secondary antibody reactive against the primary antibody species was generated and conjugated to a detectable marker (eg, horseradish peroxidase, HRP, or diaminobenzedine, DAB). An alternative method that can be used is polyclonal mirror imaging complementary antibodies or polyMICATM (Polyclonal Mirror Image Complementary Antibodies; The Binding Site Limited, Birmingham, UK; Mangham, DC et al. (1999) "Novel Immunohistochemical Detection System Using Mirror Image Complementary Antibodies (MICA),” Histopathology 35(2):129-33). The PolyMICATM technique can be used to test the binding of primary antibodies (eg anti-B7-H3 antibodies) to normal and cancerous tissue. Several types of polyMICA™ Detection kits are commercially available: Product no. HK004.D is a polyMICATM Detection kit that uses DAB chromogen; product no. HK004.A is a polyMICATM Detection kit that uses AEC chromogen. Alternatively, the primary antibody can be directly labeled with the detectable marker...
[00148] The first step in IHC screening to select an appropriate antibody is the binding of elevated primary antibodies in mice (eg anti-B7-H3 antibodies) to one or more immunogens (eg cell or tissue samples) . In one embodiment, the tissue samples are frozen tissue sections from different organs. Cell or tissue samples can be cancerous or non-cancerous.
[00149] Frozen tissues can be prepared, sectioned, with or without fixation, and IHC performed by any of a variety of methods known to one familiar with the technique (see, for example, Stephan et al. (1999) “Distribution And Function Of The Adhesion Molecule BEN During Rat Development," Dev. Biol. 212:264-277 and Stephan et al. (1999) "Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation," Endocrinology 140:5841-5854). V. METHODS OF CHARACTERIZATION OF ANTI-B7-H3 ANTIBODIES
[00150] Any of several methods can be used to characterize anti-B7-H3 antibodies. One method is to identify the epitope it attaches to. Epitope mapping is commercially available from various sources, for example, Pepscan Systems (Lelystad, The Netherlands). Epitope mapping can be used to determine the sequence to which an anti-B7-H3 antibody binds. The epitope can be a linear epitope, that is, contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch.
Peptides of varying lengths (eg preferably at least 4 to 6 amino acids in length) can be isolated or synthesized (eg recombinantly) and used to bind assays with the anti-B7-H3 antibody. The epitope to which the anti-B7-H3 antibody binds can be determined in a systematic screen using overlapping peptides derived from the extracellular sequence and determining binding by the anti-B7-H3 antibody.
[00152] Yet another method that can be used to characterize an anti-B7-H3 antibody is to use competition assays with other antibodies known to bind to the same antigen, ie, B7-H3 to determine whether anti-B7- H3 binds to the same epitope as the other antibodies. Examples of commercially available antibodies to B7-H3 may be available and can be identified using the binding assays taught herein. Competition trials are well known to those skilled in the art, and such procedures and illustrative data are further detailed in the Examples. Anti-B7-H3 antibodies can be further characterized by the tissues, type of cancer or type of tumor to which they attach.
[00153] Another method of characterizing anti-B7-H3 antibodies is by the antigen to which they bind. Anti-B7-H3 antibodies have been used in Western blots with cell lysates from various human cancers. As is known to one skilled in the art, Western blotting can involve continuous cell lysates and/or cell fractions in a denaturing or non-denaturing gel, transferring proteins to nitrocellulose paper, and then probing the stain with an antibody (for example , anti-B7-H3 antibody) to see which proteins are bound by the antibody. B7-H3 is associated with a number of human cancers from different tissues including, but not limited to, cervix, breast, ovary, pancreas and lung. SAW. CANCER DIAGNOSTIC METHODS USING ANTI-B7-H3 ANTIBODIES AND B7-H3 MODULATORS
Monoclonal antibodies to B7-H3 made by the methods described herein can be used to identify the presence or absence of cancer cells in a variety of tissues, including, but not limited to, ovary, breast, lung, prostate, colon, kidney, pancreas, skin, thyroid, brain, heart, liver, stomach, nerve, blood vessels, bone, and upper digestive tract. Monoclonal antibodies to B7-H3 made by the methods described here can also be used to identify the presence or absence of cancer cells, or their level, that are circulating in the blood after their release from a solid tumor. Such a circulating antigen may be an intact B7-H3 antigen, or its fragment which retains the ability to be detected according to the methods taught here. Such detection can be accomplished by FACS analysis using standard methods commonly used in the art.
[00155] These uses may involve the formation of a complex between B7-H3 and an antibody that specifically binds to B7-H3. Examples of such antibodies include, but are not limited to, those anti-B7-H3 monoclonal antibodies produced by hybridomas BRCA84D, BRCA69D, and PRCA157. The formation of such a complex can be in vitro or in vivo. Without being bound by theory, the anti-B7-H3 monoclonal antibody can bind to B7-H3 through the extracellular domain of B7-H3 and can then be internalized.
[00156] In a preferred embodiment of the diagnostic methods of this invention, the antibody carries a detectable label. Examples of labels that can be used include a radioactive agent or a fluorophore, such as phycoerythrin isothiocyanate or fluorescein (also known as fluoroisothiocyanate or FITC).
[00157] As with other known antibodies used commercially for diagnostic and therapeutic purposes, the target antigen of this invention is widely expressed in normal tissue. It is also regulated in some tumors. Therefore, the particular dosages and routes of administration of the antibodies of this invention, as used for diagnostics or therapeutic agents, will be tailored to the particular tumor or disease state in question, as well as the particular individual being treated.
[00158] One method of using the antibodies for diagnosis is imaging the tumor in vivo by binding the antibody to a radioactive or radio-opaque agent, administering to the individual and using an X-ray machine or other imaging machine to visualize the location of the tumor. antibody labeled on the surface of cancer cells expressing the antigen. Antibody is administered at a concentration that promotes binding under physiological conditions.
[00159] In vitro techniques for detecting B7-H3 are routine in the art and include enzyme-linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.
[00160] In aspects of this invention, methods of radiologically imaging tumors or neoplasms, or measuring the effectiveness of a method of treatment with a radiolabelled antibody, comprising the step of administering a radiolabeled tumor-specific antibody to an individual following the practice of this invention. The radiolabelled antibody may be a monoclonal or polyclonal antibody comprising a radiolabel, preferably selected from the group consisting of Technetium-99m, Indium-111, Iodine-131, Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177, Copper- 64, Scandium-47, Yttrium-90. Monoclonal antibodies labeled with therapeutic radionuclides such as Iodine-131, Rhenium-188, Holmium-166, Samarium-153 and Scandium-47, which do not compromise antibody immunoreactivity and are not broken down in vivo, are especially preferred. The person skilled in the art will appreciate that other radioactive isotopes are known, and may be suitable for specific applications. Radiological imaging can be conducted using Single Photon Emission Computer Tomography (SPECT), Positron Emission Tomography (PET), Computed Tomography (CT - Computer Tomography) or Nuclear Resonance Imaging (MRI - Magnetic Resonance Imaging). Correlative imaging, which allows for greater anatomical definition of the location of metastases located by radioimmunoassay imaging, is also contemplated.
[00161] In other methods, cancer cells are removed and tissue prepared by immunohistochemistry by methods well known in the art (eg integration into a freezing, freezing and sectioning compound, with or without fixation; fixation and integrated paraffin with or without various antigen retrieval and counterstaining methods). Monoclonal antibodies can also be used to identify cancer cells at different stages of development. Antibodies can also be used to determine which individuals' tumors express the antigen on their surface at a predetermined level and are thus candidates for immunotherapy using antibodies directed against said antigen. Antibodies can recognize both primary and metastatic cancers that express B7-H3. As used herein, detection can include qualitative and/or quantitative detection, and can include comparing the level measured in a normal cell to increased level of expression of B7-H3 in cancer cells.
[00162] The invention also provides methods to aid in diagnosing cancer characterized by cancer cells expressing B7-H3 in an individual using any antibody that binds to B7-H3 and any other methods that can be used to determine the level of expression of B7-H3. As used herein, methods for “aiding diagnosis” mean that these methods help to make a clinical determination regarding the classification, or nature, of the cancer, and may or may not be conclusive regarding the definitive diagnosis. Consequently, a method to aid in the diagnosis of cancer may comprise the step of detecting the level of B7-H3 in a biological sample from the individual and/or determining the level of expression of B7-H3 in the sample. Antibodies that recognize the antigen, or its portion, can also be used to create diagnostic immunoassays to detect the antigen released or secreted from live or dead cancer cells in bodily fluids, including, but not limited to, blood, saliva, urine, fluid pulmonary, or ascitic fluid.
[00163] Not all cells in a tumor of particular interest will express B7-H3, and cancer cells in other tissues can express B7-H3, so an individual should be screened for the presence or absence of B7-H3 in cancer cells for determine the usefulness of immunotherapy in the individual. Anti-B7-H3 antibodies made by the methods described herein can be used to determine whether an individual diagnosed with cancer can be considered a candidate for immunotherapy using antibodies directed against B7-H3. In one embodiment, a cancerous tumor or biopsy sample can be tested for expression of B7-H3 using antibodies directed against B7-H3. Individuals with cancer cells expressing B7-H3 are suitable candidates for immunotherapy using antibodies directed against B7-H3. Anti-B7-H3 antibody staining can also be used to distinguish cancerous tissue from normal tissue.
[00164] Methods of using anti-B7-H3 antibodies for diagnostic purposes are useful both before and after any form of anti-cancer treatment, eg chemotherapy or radiotherapy, to determine which tumors are most likely to respond to a given treatment , prognosis for individual with cancer, tumor subtype or origin of metastatic disease, and disease progression or response to treatment.
The compositions of this invention are also suitable for diagnosing disease states other than cancer, using the methods generally described above in application with other diseased (non-cancerous) cells. Disease states suitable for use in the methods of this invention include, but are not limited to, diseases or disorders associated with inflammatory or autoimmune responses in individuals. The methods described above can be used to modulate inflammatory or autoimmune responses in individuals. Diseases and conditions resulting from inflammation and autoimmune disorders that can be diagnosed and/or treated using the compositions and methods of the invention include, by way of illustration and not limitation, multiple sclerosis, meningitis, encephalitis, stroke, other trauma cerebral, inflammatory bowel disease including ulcerative colitis and Crohn's disease, myasthenia gravis, lupus, rheumatoid arthritis, asthma, acute juvenile diabetes, AIDS dementia, atherosclerosis, nephritis, retinitis, atopic dermatitis, psoriasis, myocardial ischemia and mediated lung injury by acute leukocyte.
[00166] Still other indications for diagnostic and/or therapeutic use of antibodies and other therapeutic agents of the invention include administration to individuals at risk of organ or graft rejection. In recent years, there has been a considerable improvement in the efficiency of surgical techniques for transplanting tissues and organs such as skin, kidney, liver, heart, lung, pancreas and bone marrow. Perhaps the main notable problem is the lack of satisfactory agents to induce immunotolerance in the recipient for the transplanted organ or allograft. When allogeneic cells or organs are transplanted into a host (ie, the donor and recipient are different individuals of the same species), the host's immune system is likely to mount an immune response to external antigens in the transplant (host versus graft disease ) leading to the destruction of the transplanted tissue.
The uses described elsewhere in this application for anti-B7-H3 antibodies also encompass the use of other agonists, antagonists and modulators of B7-H3 as described herein. In such embodiments, the agonist, antagonist or other non-antibody modulator of B7-H3 is substituted for the antibody B7-H3 in the steps described, and changes within the scope of the ordinarily skilled artisan are made to adapt the method to the modulatory composition of B7 -H3 replaced.
[00168] Monoclonal antibodies to B7-H3 made by the methods described herein can be used to identify the presence or absence of human cancer stem cells in a variety of tissues. Cancer stem cells (CSCs) have been hypothesized to play a role in tumor growth and metastasis (Ghotra, VP et al. (2009) “The Cancer Stem Cell Microenvironment And Anti-Cancer Therapy” Int. J. Radiat. Biol. 85(11):955-962; Gupta, PB et al. (2009) "Cancer Stem Cells: Mirage Or Reality " Nat. Med. 15(9): 1010-1012; Lawson, JC et al.( 2009) “Cancer Stem Cells In Breast Cancer And Metastase” Breast Cancer Res. Treat. 118(2):241-254; Hermann, PC et al. (2009) “Pancreatic Cancer Stem Cells—Insights And Perspectives” Expert Opin. Biol Ther. 9(10):1271-1278; Schatton, T. et al. (2009) "Identification And Targeting Of Cancer Stem Cells," Bioessays 31(10):1038-1049; Mittal, S. et al. 2009) “Cancer Stem Cells: The Other Face Of Janus,” Amer. J. Med. Sci. 338(2):107-112; Alison, MR et al. (2009) “Stem Cells And Lung Cancer: Future Therapeutic Targets ” Expert Opin. Biol. Ther. 9(9):1127-1141; Charafe-Jauffret, E. et al. (2009) “Breast Cancer Stem Cell s: Tools And Models To Rely On,” BMC Cancer 9:202; Scopelliti, A. et al. (2009) “Therapeutic Implications Of Cancer Initiating Cells,” Expert Opin. Biol. The R. 9(8): 1005-1016; PCT Publication WO 2008/091908). Under this hypothesis, CSCs provide a subset of distinct, small cells within each tumor that are capable of indeterminate self-regeneration and development into more adult tumor cell(s) that are relatively limited in their ability to replicate. It has been hypothesized that these cancer stem cells may be more resistant to chemotherapeutic agents, radiation or other toxic conditions, and thus, persist after clinical therapies and further grow into secondary tumors, metastasize or be responsible for relapse. It has been suggested that CSCs may elevate stem cells from “normal” tissue or from progenitor cells from more differentiated tissue.
[00169] Human cancer stem cells have several defining characteristics. Such features are described in PCT Publication WO 2008/091908 and are incorporated herein by reference. Monoclonal antibodies to cell surface targets on cancer stem cells can be used to identify the presence or absence of cancer stem cells in a variety of tissues. Monoclonal antibodies to B7-H3 made by the methods described here can also be used to identify the presence or absence of cancer stem cells, or the level of cancer stem cells in a sample or tissue or circulating after their release from a solid tumor. Such a circulating antigen may be an intact B7-H3 antigen, or a fragment thereof that retains the ability to be detected according to the methods taught here. Such detection can be performed by FACS analysis using standard methods commonly used in the art. In another embodiment, such detection can be accomplished by immunohistochemical analysis of tissue samples using standard methods commonly used in the art.
[00170] These uses may involve the formation of a complex between B7-H3 and an antibody that specifically binds to B7-H3 on cancer stem cells. Examples of such antibodies include, but are not limited to, those anti-B7-H3 monoclonal antibodies produced by hybridomas BRCA84D, BRCA69D, and PRCA157. The formation of such a complex can be in vitro or in vivo.
[00171] The uses described in this application claiming its use for anti-B7-H3 antibodies also encompass the use of other B7-H3 agonists, antagonists and modulators as described herein for use in the identification and treatment of cancer stem cells . In such embodiments, anti-B7-H3 antibodies and other B7-H3 agonists, antagonists and modulators are used for identification, diagnosis or therapeutic treatment of cancer stem cells using methods similar to those described, and alterations within the scope of the ordinary skilled person are made to adapt the method for the identification/diagnosis or treatment of cancer stem cells. VII. PREFERRED COMPOSITIONS OF THE PRESENT INVENTION
The present invention encompasses compositions, including pharmaceutical compositions, comprising anti-B7-H3 antibodies, polypeptides derived from anti-B7-H3 antibodies, polynucleotides comprising sequence encoding anti-B7-H3 antibodies, and other agents as described herein. As used herein, the compositions further comprise one or more antibodies, polypeptides and/or proteins that bind to B7-H3, agonists, antagonists, B7-H3 modulators, and/or one or more polynucleotides comprising sequences that encode one or more antibodies, polypeptides and proteins that bind to B7-H3.
The invention further provides for conjugates of any B7-H3 peptide agonist, antagonist or modulator, and additional chemical structures that support the intended function or functions of the particular B7-H3 peptide agonist, antagonist or modulator.
Such conjugates include agonist, antagonist or modulator of B7-H3 peptide covalently linked to a macromolecule such as any solid, insoluble support matrix used in the diagnostic, screening or purification procedures discussed herein. Suitable matrix materials include any substance that is chemically inert, has high porosity, and has large numbers of functional groups capable of forming covalent bonds with peptide linkers. Examples of matrix materials and procedure for preparing linker conjugates are described in Dean et al. (Eds) AFFINITY CHROMATOGRAPHY: A PRACTICAL APPROACH, IRL Press (1985); Lowe, “Introduction to Affinity Chromatography”, EM Work et al. (eds) LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY, Vol. 7, Part II, North-Holland (1979); Porath et al, “Biospecific Affinity Chromatography”, in Neurath, H. et al. (eds), AS PROTEINS, 3rd ed., Vol. 1, pp. 95-178 (1975); and Schott, H. AFFINITY CHROMATOGRAPHY, Macel Dekker, Inc. NY (1984).
Also provided herein are conjugates of agonist, antagonist or modulator of B7-H3 peptide and any reporter moiety used in the diagnostic procedures discussed herein. The B7-H3 peptide agonist, antagonist or modulator agents, polypeptides and proteins of this invention, including anti-B7-H3 antibodies, are further identified and characterized by any (one or more) of the following criteria: (a) an ability to specifically bind to B7-H3 (and in particular B7-H3 molecules that are expressed on the surfaces of cancer cells, including, but not limited to, kidney, prostate, or lung cancer cells); (b) an ability to competitively inhibit preferential binding of a known anti-B7-H3 antibody to B7-H3, including the ability to preferentially bind to the same epitope of B7-H3 to which the original antibody preferentially binds; (c) an ability to bind to a moiety of B7-H3 that is exposed on the surface of a living cell in vitro or in vivo; (d) an ability to bind to a portion of B7-H3 that is exposed on the surface of live cancer cells that express B7-H3; (e) an ability to release a chemotherapeutic agent into cancer cells (such as kidney, prostate, or lung cancer cells) expressing B7-H3 on their surface; and/or(f) an ability to release a therapeutic agent or detectable marker in cancer cells (such as, but not limited to, prostate cancer cells) expressing B7-H3 on their surface.
A preferred antibody of the invention will exhibit differential IHC staining relative to normal, non-cancerous tissue and, in addition, will be able to test in primate (and particularly crab monkey) models of antibody efficacy. Preferred antibodies of the present invention will additionally exhibit desirable levels of antigen specificity and affinity. Preferred antibodies of the present invention will additionally exhibit desirable levels of immunomodulatory activity and cellular internalization.
In some embodiments, the antibody of the invention is an antibody that is produced by hybridoma BRCA84D, BRCA69D, or PRCA157, or their progeny. The present invention also encompasses various antibody formulations produced by these deposited hybridomas and equivalent antibodies or polypeptide fragments (e.g., Fab, Fab", F(ab")2 Fv, Fc, etc.), chimeric antibodies, single chain ( scFv), its mutants, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of any such antibodies or equivalents that comprise an antigen (B7-H3), recognition site of the required specificity. The invention also provides human antibodies that display one or more of the biological characteristics of an anti-B7-H3 family member. Equivalent antibodies of the anti-B7-H3 family (including humanized antibodies and human antibodies), polypeptide fragments, and polypeptides comprising any such fragment are identified and characterized by any (one or more) of the five criteria described above. Exemplary humanized and murine variable domain sequences of an anti-B7-H3 antibody are provided in PCT Publication WO 2008/066691. Such sequences are provided by way of illustration and not limitation, and different sequences, as well as fragments and variables of sequences provided, are encompassed within the scope of this invention.
BRCA84D, BRCA69D, and PRCA157 are the preferred B7-H3 antibodies of the present invention due to their clear normal tissue IHC profiles, stronger tumor/differential normal IHC, moderate to strong binding (BIACORETM)/IHC), reactivity cross-linking to B7-H3 from crab monkeys and potent activity to universal DARTTM molecules (“UDARTTMs”) relative to other antibodies. In particularly preferred embodiments, the invention encompasses chimeric and humanized variables from such preferred antibodies, as well as natural and chimeric and humanized variables from such preferred antibodies which have modified Fc regions as described below. The invention further encompasses DARTTM molecules that possess the epitope binding regions of such antibodies, particularly in conjunction with epitope binding region(s) that bind(s) to the T cell receptor, NKG2D receptor, or an antigen associated with the tumor or with a hapten such as fluorescein (for example, fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC).
In some embodiments, antibodies, polypeptides and proteins of the invention that bind to B7-H3 are antibodies, polypeptides and proteins that competitively inhibit preferential binding of an anti-B7-H3 antibody specified herein to B7-H3. In some embodiments, antibodies, polypeptides and proteins preferentially bind to the same epitope on B7-H3 as the mu-anti-B7-H3 antibody preferentially binds.
Accordingly, the invention provides any of the following (or compositions, including pharmaceutical compositions, comprising any of the following): (a) an antibody produced by the host cell with a deposit number identified above or its progeny; (b) a humanized form of such an antibody; (c) an antibody comprising one or more of the light chain and/or heavy chain variable regions of such an antibody; (d) a chimeric antibody comprising variable regions homologous or derived from variable regions of a heavy chain and a light chain of such an antibody, and constant regions homologous or derived from constant regions of a heavy chain and a light chain of a human antibody; (e) an antibody comprising one or more of the light chain and/or heavy chain CDRs (at least one, two, three, four, five, or six) of such an antibody; (f) an antibody comprising a light and/or a heavy chain of such an antibody; (g) a human antibody that is equivalent to such an antibody. A humanized form of the antibody may or may not have CDRs identical to the original antibody, or antibody produced by a host cell with a deposit number identified above. Determining the CDR regions is well within the skill of the art. In some embodiments, the invention provides an antibody that comprises at least one CDR that is substantially homologous to at least one CDR, at least two, at least three, at least four, at least 5 CDRs of an antibody produced by one of the deposited hybridomas. identified above (or, in some embodiments substantially homologous to all 6 CDRs of one such antibody, or derived from one such antibody), or antibody produced by the host cell with a deposit number identified above. Other embodiments include antibodies having at least two, three, four, five or six CDR(s) that are substantially homologous to at least two, three, four, five or six CDRs of an antibody produced from a deposited hybridoma as identified herein. or derivatives of such an antibody. It is understood that, for the purposes of this invention, the binding specificity and/or overall activity (which may be in terms of releasing a chemotherapeutic agent to or in cancer cells to reduce the growth and/or proliferation of cancer cells, to induce apoptotic cell death in the cancer cell, to delay metastasis development, and/or palliative treatment) is generally withheld, although the extent of activity may vary compared to an antibody produced by a deposited hybridoma (may be larger or smaller). The invention also provides methods of making any of these antibodies. Antibody manufacturing methods are known in the art and are described herein.
The invention also provides polypeptides comprising an amino acid sequence of the antibodies of the invention. In some embodiments, the polypeptide comprises one or more variable regions of the antibody light chain and/or heavy chain. In some embodiments, the polypeptide comprises one or more of the antibody's light chain and/or heavy chain CDRs. In some embodiments, the polypeptide comprises three CDRs from the antibody light chain and/or heavy chain. In some embodiments, the polypeptide comprises an antibody amino acid sequence that has any of the following: at least 5 contiguous amino acids from an original antibody sequence, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids, wherein at least 3 of the amino acids are from an antibody variable region. In one embodiment, the variable region is from a light chain of the original antibody. In another embodiment, the variable region is from an antibody heavy chain. In another embodiment, the 5 (or more) contiguous amino acids are from a complementarity determining region (CDR) of the antibody.
In some embodiments of this invention, cells of this invention that express B7-H3, a portion of B7-H3, anti-B7-H3 antibodies or other B7-H3 binding polypeptides of this invention are administered directly to an individual to modulate biological activity of B7-H3 in vivo.
The preferred anti-B7-H3 antibodies of the present invention are BRCA84D, BRCA69D and PRCA157, all such antibodies are murine antibodies to the human B7-H3 molecule. The amino acid and polynucleotide sequences encoding the variable light chain and variable heavy chain of BRCA84D, BRCA69D, and PRCA157 are shown below along with the respective CDR1, CDR2 and CDR3 domains of each chain. Those skilled in the art, therefore, will be able to construct antibodies that have such CDRs, as well as their derivatives, capable of binding to epitopes by BRCA84D, BRCA69D and PRCA157. A. BRCA84D SEQUENCES (1) BRCA84D LIGHT CHAIN SEQUENCES
BRCA84D Variable Light Chain Amino Acid Sequence (SEQ ID NO:3): DIAMTQSQKF M S T V GDRV S VTCKASQNVD INVAWY QQK P GO £PK ALIY S ASYRYSGVPD RFTGSGSGTD FTLTINNVQS EDLYKTFFCQQ GT YW
[00185] Polynucleotide Sequence Encoding Light Chain Variable BRCA84D (SEQ ID NO: 4): gacattgcga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc gtcacctgca aggccagtca gaatgtggat actaatgtag cctggtatca acagaaacca gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaacaa tgtgcagtct gaagacttgg cagagtattt ctgtcagcaa tataacaact atccattcac gttcggctcg gggacaaagt tggaaaaaa
[00186] BRCA84D Variable Light Chain CDR1 (SEQ ID NO:5):

Polynucleotide Sequence Encoding Variable Light Chain CDR1 BRCA84D (SEQ ID NO:6): aaggccagte agaatgtgga tactaatgta gee
BRCA84D Variable Light Chain CDR2 (SEQ ID NO:7):

[00189] Polynucleotide Sequence Encoding BRCA84D Variable Light Chain CDR2 (SEQ ID NO:8): tcggcatcct accggtacag t.
[00190] BRCA84D Variable Light Chain CDR3 (SEQ ID NO: 9):

[00191] Polynucleotide Sequence Encoding Variable Light Chain CDR3 BRCA84D (SEQ ID NO:10): cagcaatat.a acaactatcc attcacg (2) BRCA84D HEAVY CHAIN SEQUENCES
[00192] Variable Heavy Chain Amino Acid Sequence BRCA84D (SEQ ID NO:11) : DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY ISSDSSAIYY ADTVKGRFTI SRDWPKNTLF LQNTSLRGRTTV TAMYYWG
[00193] Polynucleotide sequence encoding heavy chain variable BRCA84D (SEQ ID NO: 12): gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc tcctgtgcag cctctggatx cactttcagt agctttggaa tgcactgggt tcgtcaggct ccagagaagg ggctggagtg ggtcgcatac attagtagtg acagtagtgc catctactat gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgg aagagggagg gaaaaeattt actaeggtag taggcttgac tactggggcô aaggcaeaac -tatcacagti; tcctca
[00194] BRCA84D Variable Heavy Chain CDR1 (SEQ ID NO:13):

[00195] Polynucleotide Sequence Encoding Variable Heavy Chain CDR1 BRCA84D (SEQ ID NO:14): tttggaatgcac
BRCA84D Variable Heavy Chain CDR2 (SEQ ID NO:15):

[00197] Polynucleotide Sequence Encoding Variable Heavy Chain CDR2 BRCA84D (SEQ ID NO:16): tacattagta gtgacagtag tgccatctac tatgcagaca cagtgaag
BRCA84D(SEQ ID NO:17) Variable Heavy Chain CDR3:

[00199] Polynucleotide Sequence Encoding Variable Heavy Chain CDR3 BRCA84D (SEQ ID NO:18): ggggagggaaa acatttacta cggtagtagg cttgactac B. BRCA69D SEQUENCES (1) BRCA69D LIGHT CHAIN SEQUENCES
[00200] Variable Light Chain Amino Acid Sequence BRCA69D (SEQ ID NO:19): DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY TSRLHSGVPS RFSGSGSGTD YSLTIDNLEQ EDIATYFCQQ GNTLPPKTFGG GTKLE
[00201] Polynucleotide Sequence Encoding Light Chain Variable BRCA69D (SEQ ID NO: 20): gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcaca atcagttgca gggcaagtca ggacattagt aattatttaa actggtatca gcagaaaaca gatggaactg ttaaactcct gatctactac acatcacgat tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat tattctctca ccattgacaa cctggagcaa gaagatattg ccacttactt ttgccaacag ggtaatacgc ttcctccgac gttcggtgga ggcaccaaac tggaaatcaa a
BRCA69D Variable Light Chain CDR1 (SEQ ID NO:21):

Polynucleotide Sequence Encoding Variable Light Chain CDR1 BRCA69D (SEQ ID NO:22): agggcaagtc aggacattag taattattta aac
BRCA69D Variable Light Chain CDR2 (SEQ ID NO:23):

Polynucleotide Sequence Encoding Light Chain CDR2 BRCA69D (SEQ ID NO:24): tacacatcac gattacactc a
BRCA69D Variable Light Chain CDR3 (SEQ ID NO:25):

Polynucleotide Sequence Encoding Variable Light Chain CDR3 BRCA69D (SEQ ID NO:26): caacagggta atacgcttcc tccgacg (2) BRCA69D HEAVY CHAIN SEQUENCES
[00208] Variable Heavy Chain Amino Acid Sequence BRCA69D (SEQ ID NO:27): QVQLQQSGAE LARPGASVKL SCKASGYTFT SYWMQWVKQR PGQGLEWIGT IYPGDGDTRY TQKFKGKATL S TADKSSSTAY MQLSSCARGYTFT SYWMQWVKQR
[00209] Polynucleotide sequence encoding BRCA69D variable heavy chain (SEQ ID NO: 28): caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagttg tcctgaaagg cttctggcta cacctttact agctactgga tgcagtgggt aaaacagagg cctggacagg gtctggaatg gattgggact atttatcctg gagatggtga tactaggtac actcagaagt tcaagcgcaa ggccacat-g act.gçagata aatctztccaα cacagccΣac atgcaactca gcagcttggc atctgaggac tútgcggtct attactgtgc aagaagaggg attccacggc tttggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca
BRCA69D Variable Heavy Chain CDR1 (SEQ ID NO:29):

Polynucleotide Sequence Encoding Variable Heavy Chain CDRi BRCA69D (SEQ ID NO:30): agctactgga tgcag
[00212] Variable Heavy Chain CDR2 BRCA69D (SEQ ID NO:31):

Polynucleotide Sequence Encoding Variable Heavy Chain CDR2 BRCA69D (SEQ ID NO:32): actatttatc ctggagatgg tgatactagg tacactcag aagttcaagg gc
BRCA69D Variable Heavy Chain CDR3 (SEQ ID NO:33):

Polynucleotide Sequence Encoding Variable Heavy Chain CDR3 BRCA69D (SEQ ID NO:34): agagggattc cacggctttg gtacttcgat gtc C. SEQUENCES OF PRCA157 (1) PRCA157 LIGHT CHAIN SEQUENCES
PRCA157 Variable Light Chain Amino Acid Sequence PRCA157 (SEQ ID NO:35): DTQMTQSFAS LSVSVGETVT ITCRSESIY SYLAWYQQKQ GKSPQLLVYN TKTLPEGVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYCQI HYGGTPWTF
[00217] Polynucleotide sequence encoding PRCA157 Variable Light Chain (SEQ ID NO: 36): gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga aactgtcacc attacatgtc gagcaagtga gagtatttac agttatttag catggtatca gcagaaacag ggaaaatctc ctcagctcct ggtctataat acaaaaacct aggttcagtg gcagtggatc aggcacacag taccagaggg tgtgccatca ttt.tct.ctga agatcaacag cctgcagcct gaagattttg ggagatatta ctgtcaacat cattatggta ctcctccgtg gacgttcggt ggaggcacca acctggaaat caaa
[00218] PRCA157 Variable Light Chain CDR1 (SEQ ID NO:37):

[00219] Polynucleotide Sequence Encoding Variable Light Chain CDR1 PRCA157 (SEQ ID NO:38): cgagcaagtg agagtattta cagttattta gca
[00220] PRCA157 Variable Light Chain CDR2 (SEQ ID NO:39):

[00221] Polynucleotide Sequence Encoding Variable Light Chain CDR2 PRCA157 (SEQ ID NO:40): aatacaaaaa ccttaccaga g
PRCA157 Variable Light Chain CDR3 (SEQ ID NO:41):

[00223] Polynucleotide Sequence Encoding Variable Light Chain CDR3 PRCA157 (SEQ ID NO:42): caacacatt atggtactcc tccgtgg (2) PRCA157 HEAVY CHAIN SEQUENCES
[00224] Variable Heavy Chain Amino Acid Sequence PRCA157 (SEQ ID NO:43): EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT PDKRLEWVAT INSGGSNTYY PDSLKGRFTI SRDMAKNTLY LQMRSLKSED TAMYYCARHD VGG
[00225] Polynucleotide sequence encoding heavy chain variable PRCA157 (SEQ ID NO: 44): gaggtgcagc aggtggagtc ggggggagac ttagtgaagc ctggagggtc cctgaaactc tcctgtgcag cctctggatt cactttcagt tcctatggca tg tc11ggg t tcgccaga ct cc to GACA aga ggctggagtg GGT CGC ACC attaatagtg gtggaagtaa eacctactat ccagaeagtt tgaaggggcg 11 cac ca tct cc ag aga caat gc caa ga acaccc 111 acctg ca aatgc gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacatgac gggggagcta tggactactg gggtcaagga acctcagtca ccgtctcctc a
[00226] Variable Heavy Chain CDR1 PRCA157 (SEQ ID NO:45):

[00227] Polynucleotide Sequence Encoding Variable Heavy Chain CDR1 PRCA157 (SEQ ID NO:46): tccatggca tgtct
[00228] Variable Heavy Chain CDR2 PRCA157 (SEQ ID NO:47):

[00229] Polynucleotide Sequence Encoding Variable Heavy Chain CDR2 PRCA157 (SEQ ID NO:48): gtcgcaacca ttaatagtgg tggaagtaac acctactatc cagacagttt gaagggg
[00230] PRCA157 Variable Heavy Chain CDR3 (SEQ ID NO:49):

[00231] Polynucleotide Sequence Encoding Variable Heavy Chain CDR3 PRCA157 (SEQ ID NO:50): catgacgggg gagctatgga ctac D. B7-H3 ANTIBODIES DESIGNED BY FC
[00232] In traditional immune function, the interaction of antibody-antigen complexes with immune system cells results in a wide variety of responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and phagocytosis to such immunomodulatory signals as regulating lymphocyte proliferation and antibody secretion. All of these interactions are initiated through the binding of the Fc domain of antibodies or immune complexes to specialized cell receptors on hematopoietic cells. The diversity of cellular responses triggered by antibodies and immune complexes results from the structural heterogeneity of the three Fc receptors: FcRI (CD64), FcRII (CD32), and FcRIII (CD 16). FcRI (CD64), FcRIIA (CD32A) and FcRIII (CD 16) are activating receptors (ie, enhancement of the immune system); FcyRIIB (CD32B) is an inhibiting (ie attenuating) immune system receptor. The amino acid sequence of the Fc region of IgG1 Fc is shown below (as SEQ ID NO:51, numbered according to Kabat et al, SEQUENCE OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, NIH, MD (1991), expressly incorporated by reference herein, and hereinafter referred to as "Kabat I"): SEQ ID N0: 51PAPELLGGPS230 VFLFPPKFKD 240 TLMISRTPEV 250 TCVWDVSHE260 DPEVKFNWYV 270DGVEVHNAKT230 KPREEQYNST 290 YRWSVLTVL 300 HQDWLNGKEY 310 KCKVSNKALP 32CAFIEKTlSKft330 KGQFREFQVY 340 TLFFSREEMT350 KNQVSLTCLV 360 KGFYFSDIftV 370EWESNGQFEM 380 NYKTTFFVLD 390 SDGSFFLYSK 0 0 LTVDKSRWQQ410 GNVFSCSVMH 420EALHNHYTQK SLSLSPGK 4.30 440
Residues 230 to 341 are the Fc CH2 region. Residues 342 to 447 are the Fc CH3 region.
The present invention includes antibodies that specifically bind to B7-H3 which comprises a variable Fc region having one or more amino acid modification (e.g. substitutions, deletions, insertions) in one or more moieties, modifications that increase affinity and variable Fc region avidity for an FcR (including activating and inhibitory FcRs). In some embodiments, said one or more amino acid modifications increase the affinity of the variable Fc region for FcRIIIA and/or FcRIIA. In another embodiment, the variable Fc region still specifically binds FcRIIB with a lower affinity than the Fc region of the comparable major antibody (i.e., an antibody that has the same amino acid sequence as the antibody of the invention except for one or more plus amino acid modifications in the Fc region). In some embodiments, such modifications increase the affinity of the variable Fc region for FcRIIIA and/or FcRIIA and also increase the affinity of the variable Fc region for FcRIIB relative to the main antibody. In other embodiments, said one or more amino acid modifications increase the affinity of the variable Fc region for FcRIIIA and/or FcRIIA but do not change the affinity of the variable Fc region for FcRIIB relative to the Fc region of the main antibody. In another embodiment, said one or more amino acid modifications increase the affinity of the variable Fc region for FcRIIIA and FcRIIA, but reduce the affinity for FcRIIB relative to the main antibody. Affinity and/or avidity results in detectable binding to FcR or FcR-related activity in cells expressing low levels of the FcR when major molecule binding activity (without the modified Fc region) cannot be detected in the cells. In other embodiments, the modified molecule exhibits detectable binding in cells expressing non-FcR receptor target antigens at a density of 30,000 to 20,000 molecules/cell, at a density of 20,000 to 10,000 molecules/cell, at a density of 10,000 to 5,000 molecules/cell , at a density of 5,000 to 1,000 molecules/cell, at a density of 1,000 to 200 molecules/cell, or at a density of 200 molecules/cell or less (but at least 10, 50, 100 or 150 molecules/cell) .
[00235] In another embodiment, said one or more modifications to the amino acids of the Fc region reduce the affinity and avidity of the antibody for one or more FcR receptors. In a specific embodiment, the invention encompasses antibodies comprising a variable Fc region, wherein said variable Fc region comprises at least one amino acid modification with respect to a wild-type Fc region, variable Fc region which only binds to an FcR, in that said FcyR is FcyRIIIA. In another specific embodiment, the invention encompasses antibodies comprising a variable Fc region, wherein said variable Fc region comprises at least one amino acid modification with respect to wild-type Fc region, variable Fc region which only binds to an FcR, wherein the said FcyR is FcyRIIA...
[00236] Preferably, the binding properties of the molecules of the invention are characterized by in vitro functional assays to determine one or more FcR mediator effector cell functions (See Section 5.2.7). The affinities and binding properties of molecules, eg antibodies, of the invention for an FcR can be determined using in vitro assays (biochemical or immunologically based assays) known in the art to determine antibody-antigen or Fc-FcR interactions, that is, specific binding of an antigen to an antibody or specific binding of an Fc region to an FcR, respectively, including, but not limited to, ELISA assay, surface plasma resonance assay, immunoprecipitation assays. In more preferred embodiments, molecules of the invention have similar binding properties in in vivo models (such as those described and disclosed herein) as those in in vitro based assays. However, the present invention does not exclude molecules of the invention that do not exhibit the desired phenotype in in vitro assays, but do not exhibit the desired phenotype in vivo.
[00237] In some embodiments, molecules of the invention comprising a variable Fc region comprise at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more modifications of amino acid) in the CH3 domain of the Fc region, which is defined as an extension of amino acids 342 to 447. In other embodiments, molecules of the invention comprising a variable Fc region comprise at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) in the CH2 domain of the Fc region, which is defined as an extension of amino acids 231 to 341. In some embodiments, molecules of the invention comprise at least two amino acid modifications (for example, having 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), wherein at least such modification is in the CH3 region and at least such modification is in the region CH2. The invention further encompasses amino acid modification in the joint region. In a particular embodiment, the invention encompasses amino acid modification in the CH1 domain of the Fc region, which is defined as the length of amino acids 216 to 230.
In particularly preferred embodiments, the invention encompasses molecules comprising a variable Fc region wherein said variable confers or has increased ADCC activity and/or increased binding to FcRIIA (CD32A), as measured using methods known to one skilled in the art. matter and exemplified here. The ADCC assays used in accordance with the methods of the invention can be NK dependent or macrophage dependent.
In particularly preferred embodiments, the invention encompasses molecules comprising a variable Fc region wherein said variable confers or has increased ADCC activity and/or increased binding to FcRIIIA (CD16A), as measured using methods known to one skilled in the art. technique exemplified here. The ADCC assays used in accordance with the methods of the invention can be NK dependent or macrophage dependent.
The Fc variables of the present invention can be combined with other Fc modifications, such as those described in U.S. Patent Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; and 6,737,056; in PCT Publication Nos. WO 2008/105886; WO 2008/002933; WO 2007/021841; WO 2007/106707; WO 06/088494; WO 05/115452; WO 05/110474; WO 04/1032269; and in WO 04/063351; and in Presta, L.G. et al. (2002) “Engineering Therapeutic antibodies for improved function,” Biochem. Trans. 30(4):487-490; Shields, R.L. et al. (2002) “Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity,” J. Biol. Chem. 26;277(30):26733-26740 and Shields, R.L. et al. (2001) “High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R,” J. Biol. Chem. 276(9):6591-6604). The invention encompasses combining an Fc variable of the invention with other Fc modifications to provide additive, synergistic or novel properties for the modified antibody. Preferably, the Fc variables of the invention augment the phenotype of the modification with which they are combined. For example, if an Fc variable of the invention is combined with a mutant known to bind FcRIIIA with greater affinity than a comparable wild-type Fc region; combination with a mutant of the invention results in a greater fold improvement in FcRIIIA affinity.
The invention encompasses antibodies that specifically bind to B7-H3 which comprise a variable Fc region, wherein the variable Fc region comprises at least one amino acid modification (e.g. having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has an increased effector function relative to a molecule comprising a wild-type Fc region, provided the variable Fc region does not have or is not just a substitution at any one or more of positions 243, 255,256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303,305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333,334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419,430, 434, 435, 437, 438, 439. In a specific embodiment, the invention encompasses such antibodies comprising a variable Fc region, wherein the variable Fc region comprises at least one modification amino acid tion (for example, having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region such that the molecule binds to an FcR with an altered affinity relative to a molecule comprising a wild-type Fc region, provided that the variable Fc region does not or is not just a substitution at any one or more of positions 243, 255, 258, 267, 269, 270, 276, 278 , 280, 283,285, 289, 292, 293, 294, 295, 296, 300, 303, 305, 307, 309,320, 322, 329, 332, 331, 337, 338, 340, 373, 376, 416, 419,434, 435 , 437, 438, 439 and lacks an alanine and any of positions 256, 290, 298, 312, 326, 333, 334, 359, 360, or 430; an asparagine at position 268; a glutamine at position 272; a glutamine, serine, or aspartic acid at position 286; a serine at position 290; a methionine at position 301; a methionine, glutamine, glutamic acid, or arginine at position 320; a glutamic acid at position 322; an asparagine, serine, glutamic acid, or aspartic acid at position 326; a lysine at position 330; a glutamine at position 334; a glutamic acid at position 334; a methionine at position 334; a histidine at position 334; a valine at position 334; a leucine at position 334; a glutamine at position 335; a lysine at position 335; or a threonine at position 339.
The invention also encompasses antibodies that specifically bind to B7-H3 which comprise a variable Fc region, wherein the variable Fc region comprises such antibodies comprising a variable Fc region, wherein the variable Fc region does not or is not just a substitution at any one or more of positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 320, 331, 333, 334 , 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, or 439 and does not have a histidine, glutamine, or tyrosine at position 280; a serine, glycine, threonine or tyrosine at position 290, an asparagine at position 294, a lysine at position 295; a proline at position 296; a proline, asparagine, aspartic acid, or valine at position 298; or a leucine or isoleucine at position 300. In another embodiment, the invention encompasses such antibodies comprising a variable Fc region, wherein the variable Fc region comprises at least one amino acid modification relative to the wild type Fc region, such that the molecule binds to an FcR with a reduced affinity relative to the molecule comprising a wild-type Fc region as long as the variable Fc region does not or is not just a substitution at any one or more of positions 243, 252, 254, 265, 268, 269 , 270, 278, 289, 292, 293, 294,295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335,338, 340, 373, 376, 382, 388, 389, 414, 416 , 419, 434, 435,437, 438, or 439. In yet another embodiment, the invention encompasses such antibodies comprising a variable Fc region, wherein the variable Fc region comprises at least one amino acid modification relative to the wild type Fc region, so that the molecule binds to FcR with an increased affinity with respect to the mol Ecula comprising a wild type Fc region as long as the variable Fc region does not have and is not merely a substitution at any one or more of positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430.
The invention also encompasses antibodies that specifically bind to B7-H3 which comprise a variable Fc region, wherein the variable Fc region does not or is not just a substitution at any one or more of positions 330, 243, 247, 298, 241, 240, 244, 263, 262, 235, 269, or 328 and does not have a leucine at position 243, an asparagine at position 298, a leucine at position 241, and isoleucine or an alanine at position 240, a histidine at position 244, a valine at position 330, or an isoleucine at position 328.
The invention particularly encompasses antibodies that specifically bind to B7-H3 which comprise a variable Fc region with increased effector function and/or altered affinities for activating and/or inhibitory receptors, wherein the variable Fc region comprises: (a ) any 1, 2, 3, 4, 5, or 6 of the following substitutions: S239D, S298A, A330L, I332E, E333A, or K334A; or (b) any one of the combinations of substitutions: (1) S298A, E333A, and K334A; (2) S239D and I332E; or (3) S239D, A330L and I332E.
The invention particularly encompasses antibodies that specifically bind to B7-H3 which comprises a variable Fc region with increased effector function and/or altered affinities for activation and/or inhibitory receptors, wherein the variable Fc region comprises a substitution:( 1) at position 288 with asparagine, at position 330 with serine and at position 396 with leucine; (2) at position 334 with glutamic acid, at position 359 with asparagine, and at position 366 with serine; (3) at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid; (4) at position 247 with leucine, and a substitution at position 421 with lysine; (5) at position 392 with threonine, and at position 396 with leucine; (6) at position 221 with glutamic acid, at position 270 with glutamic acid, at position 308 with alanine, at position 311 with histidine, at position 396 with leucine, and at position 402 with aspartic acid; (7) at position 419 with histidine, and a substitution at position 396 with leucine; (8) at position 240 with alanine, and at position 396 with leucine; (9) at position 410 with histidine, and at position 396 with leucine; (10) at position 243 with leucine, at position 305 with isoleucine, at position 378 with aspartic acid, at position 404 with serine, and at position 396 with leucine; (11) at position 255 with isoleucine, and at position 396 with leucine; (12) at position 370 with glutamic acid and at position 396 with leucine;(13) at position 270 with glutamic acid; or (14) any combination of the above substitutions (1) to (12).
In a specific embodiment, the invention encompasses an antibody that specifically binds to B7-H3 which comprises a variable Fc region comprising the substitution: F243L, R292P, and Y300L. In yet a specific embodiment, the invention encompasses an antibody that specifically binds to B7-H3 that comprises a variable Fc region that comprises the substitution: L235V, F243L, R292P, Y300L, and P396L. In yet a specific embodiment, the invention encompasses an antibody that specifically binds to B7-H3 that comprises a variable Fc region that comprises the substitution F243L, R292P, Y300L, V305I, and P396L.
In yet a specific embodiment, the invention encompasses an antibody that specifically binds to B7-H3 comprising a variable Fc region comprising a substitution at position 396 with leucine, at position 270 with glutamic acid and at position 243 with leucine . In another specific embodiment, the molecule further comprises one or more amino acid modifications such as those described herein.
[00248] The invention particularly encompasses antibodies that specifically bind to B7-H3 comprising a variable Fc region with increased effector function and/or altered affinities for activating and/or inhibitory receptors, which have an amino acid modification in one or more of the following positions: 119, 125, 132, 133, 141, 142,147, 149, 162, 166, 185, 192, 202, 205, 210, 214, 215, 216,217, 218, 219, 221, 222, 223, 224, 225, 227, 229, 231, 232,233, 235, 240, 241, 242, 243, 244, 246, 247, 248, 250, 251,252, 253, 254, 255, 256, 258, 261, 262, 263, 268, 269, 270,272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288, 289,290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320,323, 326, 327, 328, 330, 333, 334, 335, 337, 339, 340, 343.344, 345, 347, 348 , 352, 353, 354, 355, 358, 359, 360, 361,362, 365, 366, 367, 369, 370, 371, 372, 375, 377, 378, 379,380, 381, 382, 383, 384, 385, 386 , 387, 388, 389, 390, 392.393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 404, 406,407, 408, 409, 410, 411, 412, 414, 415, 416, 417, 419, 420.421, 422, 423, 424, 427, 428, 431, 433, 435, 436, 438, 440,441, 442, 443, 446, or 447. Preferably such mutations result in molecules that have been conferred with effector cell-mediated function and, optionally, have an altered affinity for an FcR as determined using methods described and exemplified herein and known to one skilled in the art.
[00249] The invention particularly encompasses antibodies that specifically bind to B7-H3 which comprise a variable Fc region with altered effector function and/or altered affinities for activation and/or inhibition receptors, which have: (I) an amino acid modification in one or more of the following positions: 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or 330 and more preferably one or more of the following modifications: V240A, V240I, F241L, F243L, P244H , S298N, L328I, A330V; wherein such antibodies exhibit altered effector function relative to antibodies that have a wild-type Fc region that lacks such modification; (II) an amino acid modification at one or more of the following positions: 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and more preferably one or more of the following modifications: D280H, D280Q, D280Y, K290G, K290S, K290T, K290Y, E294N, Q295K, Y296P, S298D, S298N, S298P, S298V, Y300I, Y300L ; wherein such antibodies exhibit altered effector function relative to antibodies that have a wild-type Fc region that lacks such modification; (III) an amino acid modification at one or more of the following positions: 255, 256, 258, 267, 268, 269 , 270,272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322,326, 329, 330 , 332, 331, 333, 334, 335, 337, 338, 339, 340,359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439, and more preferably one or more of the following modifications: T256A , H268N, E272Q, N286D, N286Q, N286S, K290A, K290S, S298A, R301M, D312A, K320E, K320M, K320Q, K320R, K322E, K326A, K326D, K326E, K326N, K33A, E3, A, A , K334H, K334L, K334M, K334Q, K334V, T335K, T335Q, T359A, K360A, E430A; wherein such antibodies exhibit altered effector function relative to antibodies that have a wild-type Fc region that lacks such modification; (IV) an amino acid modification at one or more of the following positions: 252, 254, 265, 268, 269, 270, 278,289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324,327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414.416, 419, 434, 435, 437, 438, or 439; wherein such antibodies exhibit reduced effector function relative to antibodies that have a wild-type Fc region that lacks such modification; (V) an amino acid modification at one or more of the following positions: 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430; wherein such antibodies exhibit increased effector function relative to antibodies which have or(VI) an amino acid modification at one or more of the following positions: R255A, T256A, E258A, S267A, H268A, H268N, E272A, E272Q, N276A, D280A, E283A , H285A, N286A,N286D, N286Q, N286S, K290A, K290S, R301M, K320E, K320M,K320Q, K320R, K322E, K326A, K326D, K326E, K326S, A330K, P331A, T335Q, S3; wherein such antibodies exhibit increased effector function relative to antibodies that have a wild-type Fc region that lacks such modification.
[00250] In other embodiments, the invention encompasses the use of any Fc variable known in the art, such as those described in Jefferis, B.J. et al. (2002) “Interaction Sites On Human IgG-Fc For FcgammaR: Current Models”, Immunol. Letter 82:57-65; Presta, L.G. et al. (2002) “Engineering Therapeutic Antibodies For Improved Function”, Biochem. Trans. 30:487-90; Idusogie, E.E. et al. (2001) “Engineered Antibodies With Increased Activity To Recruit Complement”, J. Immunol. 166:2571-75; Shields, R.L. et al. (2001) "High Resolution Mapping Of The Binding Site On Human IgG1 For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgG1 Variants With Improved Binding To The Fc gamma R", J. Biol. Chem. 276:6591-6604; Idusogie, E.E. et al. (2000) “Mapping Of The Clq Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc”, J. Immunol. 164:417884; Reddy, M.P. et al. (2000) "Elimination Of Fc Receptor Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4", J. Immunol. 164:1925-1933; Xu, D. et al. (2000) “In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies”, Cell. Immunol. 200:16-26; Armor, K.L. et al. (1999) “Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities”, Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) "Modulation Of Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions", Immunol. Letter 54:101-04; Lund, J. et al. (1996) "Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains", J. Immunol. 157:4963-4969; Hutchins et al. (1995) "Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-1H", Proc. Natl. Academic Sci. (U.S.A.) 92: 1 1980-84; Jefferis, R. et al. (1995) "Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glycosylation", Immunol. Letter 44:111-17; Lund, J. et al. (1995) "Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors", FASEB J. 9:115-19; Alegre, M.L. et al. (1994) “A Non-Activating “Humanized” Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo”, Transplantation 57:1537-1543; Lund et al. (1992) “Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma R11,” Mol. Immunol. 29:53-59; Lund et al. (1991) "Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG," J. Immunol. 147:2657-2662; Duncan, A.R. et al. (1988) “Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG,” Nature 332:563-564; U.S. Patent No. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publications WO 00/42072 and PCT WO 99/58572.
The invention encompasses molecules comprising variable Fc regions consisting of or comprising any of the mutations listed in the table below in Table 1.





In specific embodiments, the variable Fc region of such anti-B7-H3 antibodies has: (1) a leucine at position 247, a lysine at position 421 and a glutamic acid at position 270; (2) a threonine at position 392, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243;..(3) a histidine at position 419, a leucine at position 396, and a glutamic acid at position 270;(4) a histidine at position 419, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243;(5) an alanine at position 240, a leucine at position 396, and a glutamic acid at position 270;(5) 6) a lysine at position 255 and a leucine at position 396;(7) a lysine at position 255, a leucine at position 396, and a glutamic acid at position 270;(8) a lysine at position 255, a leucine at position 396, a glutamic acid at position 270, and a lysine at position 300;(9) a lysine at position 255, a leucine at position 396, a glutamic acid at position 27 0, and a glycine at position 292;(10) a lysine at position 255, a leucine at position 396, a glutamic acid at position 270, and a leucine at position 243;(11) a glutamic acid at position 370, a leucine at position 396, and a glutamic acid at position 270;(12) a glutamic acid at position 270, an aspartic acid at position 316, and a glycine at position 416;(13) a leucine at position 243, a proline at position 292 , an isoleucine at position 305, and a leucine at position 396;(14) a leucine at position 243, a glutamic acid at position 270, an asparagine at position 392 and a leucine at position 396;(15) a leucine at position 243 , a leucine at position 255, a glutamic acid at position 270 and a leucine at position 396; (16) a glutamine at position 297; or (17) any combination of substitutions (1) to (16) above.
In some embodiments, the molecules of the invention further comprise one or more glycosylation sites, such that one or more carbohydrate moieties are covalently attached to the molecule. Preferably, molecules of the invention with one or more glycosylation sites and/or one or more modifications in the Fc region confer or have an increased antibody-mediated effector function, e.g., increased ADCC activity, compared to a major antibody. In some embodiments, the invention further comprises molecules comprising one or more amino acid modifications that are directly or indirectly known to interact with a carbohydrate moiety of the antibody, including, but not limited to, amino acids at positions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301. Amino acids that directly or indirectly interact with a carbohydrate moiety of an antibody are known in the art, see, for example, Jefferis et al. ., 1995 Immunology Letters, 44:111-7, which is incorporated herein by reference in its entirety.
[00254] In another embodiment, the invention encompasses molecules that have been modified by introducing one or more glycosylation sites at one or more sites on the molecules, preferably without changing the functionality of the molecules, for example, binding the activity to the target antigen or FcyR . Glycosylation sites can be introduced into the variable and/or constant region of the molecules of the invention. As used herein, "glycosylation sites" include any amino acid sequence in an antibody to which an oligosaccharide (ie, carbohydrates containing two or more simple sugars linked together) will specifically and covalently attach. Oligosaccharide side chains are typically attached to the backbone through N or O bonds. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxyamino acid, eg serine, threonine. Molecules of the invention may comprise one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any glycosylation site for N-linked or O-linked glycosylation known in the art can be used in accordance with the immediate invention . An exemplary N-linked glycosylation site that is in accordance with those of the present invention is the amino acid sequence: Asn-X-Thr/Ser, where X can be any amino acid and Thr/Ser denoted a threonine or a serine. Such site(s) can be introduced into a molecule of the invention using methods well known in the art to which this invention belongs (see, for example, IN VITRO MUTAGENESIS, RECOMBINANT DNA: A FAST COURSE, JD Watson, et al. WH Freeman and Company , New York, 1983, chapter 8, pp. 106-116, which is incorporated herein by reference in its entirety. An exemplary method for introducing a glycosylation site into a molecule of the invention can comprise: modification or mutation of an amino acid sequence of the molecule such that the desired sequence Asn-X-Thr/Ser is obtained.
[00255] In some embodiments, the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by adding or deleting a glycosylation site. Methods for modifying the carbohydrate content of antibodies are well known in the art and encompassed within the invention, see, for example, U.S. Patent No. 6,218,149; EP 0 359 096 B1; US Publication No. US 2002/0028486; WO 03/035835; US Publication No. 2003/0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. In other embodiments, the invention encompasses methods of modifying the carbohydrate content of a molecule of the invention by excluding one or more endogenous carbohydrate portions of the molecule. In a specific embodiment, the invention encompasses changing the glycosylation site of the Fc region of an antibody by modifying the adjacent positions to 297. In a specific embodiment, the invention encompasses modifying position 296 so that position 296 and not position 297 is glycosylated.
[00256] The effector function can also be modified by techniques such as by introducing one or more cysteine residues into the Fc region, thereby allowing the formation of an interchain disulfide bond to occur in that region, resulting in the generation of a homodimeric antibody that may have improved internalization capability and/or increased complement-mediated cell killing and ADCC (Caron, PC et al. (1992) "Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies" J. Exp. Med. 176:1191- 1195; Shopes, B. (1992) "A Genetically Engineered Human IgG Mutant With Enhanced Cytolytic Activity" J. Immunol. 148(9):2918-2922 Homodimeric antibodies with increased antitumor activity can also be prepared using heterobifunctional crosslinkers as described in Wolff, EA et al.(1993) "Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice" Cancer Research 53:2560-2565. Alternatively, an antibody can be designed which has r dual Fc regions and may thus have enhanced complementary lysis and ADCC capabilities (Stevenson, G.T. et al. (1989) "A Chimeric Antibody With Dual Fc Regions (bisFabFc) Prepared By Manipulations At The IgG Hinge" Anti-Cancer Drug Design 3:219-230). E. DARTTM (DUAL Affinity Redirection Reagent) B7-H3
[00257] As discussed above, the present invention further encompasses "DARTTM" (dual affinity redirection reagent) molecules comprising at least two polypeptide chains that form at least two epitope binding sites, at least one of which specifically joins B7-H3.
In preferred embodiments, the first polypeptide chain of the DART™ comprises: (i) a domain (A) comprising a light chain variable domain binding region of a first immunoglobulin (VL1) specific for an epitope (1) ;(ii) a domain (B) comprising a binding region of a heavy chain variable domain of a second immunoglobulin (VH2) specific for an epitope (2); and (iii) a domain (C).
The second polypeptide chain of such DARTTM comprises: (i) a domain (D) comprising an epitope-specific second immunoglobulin light chain variable domain binding region (VL2) (2); (ii) a domain (E) comprising an epitope-specific first immunoglobulin (VH1) chain variable domain binding region (1); and (iii) a domain (F).
The DARTTM domains (A) and (B) do not associate with each other to form an epitope binding site. Similarly, the DARTTM domains (D) and (E) do not associate with each other to form a deepitope binding site. Instead, the DART™ domains (A) and (E) associate to form a binding site that binds to the epitope (1); said DART™ domains (B) and (D) associate to form a binding site that binds to said epitope (2). Domains (C) and (F) are covalently associated together.
[00261] Each polypeptide chain of the DARTTM molecule comprises a VL domain and a VH domain, which are covalently linked so that domains are restricted from self-assembly. The interaction of two of the polypeptide chains will produce two VL-VH pairings, forming two epitope binding sites, ie, a bivalent molecule. Neither the VH or VL domain is restricted to any position within the polypeptide chain, i.e., restricted to the amino (N) or carboxy (C) terminus, nor are the domains restricted in their relative positions to one another, i.e. , the VL domain can be an N-terminal to the VH domain and vice versa. The only restriction is that the complementary polypeptide chain is available in order to form functional DARTTMs. Where the VL and VH domains are derived from the same antibody, the two complementary polypeptide chains may be identical. For example, where the binding domains are derived from an antibody specific for epitope A (i.e., the binding domain is formed from a VLA-VHA interaction), each polypeptide will comprise a VHA and a VLA. Homodimerization of two antibody polypeptide chains will result in the formation of two VLA-HAV binding sites, resulting in a bivalent monospecific antibody. Where the VL and VH domains are derived from antibodies specific to different antigens, the formation of a functional bispecific DARTTM requires the interaction of two different polypeptide chains, that is, the formation of a heterodimer. For example, for a bispecific DART™, a polypeptide chain will comprise a VLA and a VLB; homodimerization of said strand will result in the formation of two VLA-HBV binding sites, which lack binding or unpredictable binding. In contrast, where two different polypeptide chains are free to interact, for example, in a recombinant expression system, one comprising a VLA and a HBV and the other comprising a VLB and a VHA, two different binding sites will form: VLA- VHA and VLB-HBV. For all DARTTM polypeptide chain pairs, the possible misalignment or detachment of two chains is a possibility, ie, interaction of the VL-VL or VH-VH domains; however, purification of functional diabodies is easily managed based on the immunospecificity of the properly dimerized binding site, using any affinity based method known in the art or exemplified herein, e.g., affinity chromatography.
One or more of the DART™ polypeptide chains may optionally comprise an Fc domain or portion thereof (for example, a CH2 domain, or CH3 domain). The Fc domain or portion thereof can be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the Fc domain (or portion thereof) is derived from IgG. In specific embodiments, the IgG isotype is IgG1, IgG2, IgG3 or IgG4 or its allotype. In one embodiment, the diabody molecule comprises an Fc domain, Fc domain comprising a CH2 domain and CH3 domain independently selected from any immunoglobulin isotype (i.e., an Fc domain comprising the IgG-derived CH2 domain, and the CH3-derived domain of IgE, or the CH2 domain derived from IgG1 and the CH3 domain derived from IgG2, etc.). The Fc domain can be projected onto a polypeptide chain comprising the diabody molecule of the invention at any position relative to other domains or portions of said polypeptide chain (for example, the Fc domain, or its portion can be c-terminal to both the VL and VH domains of the polypeptide chain; it can be n-terminal to both the VL and VH domains; or it can be N-terminal to one domain and c-terminal to another (i.e., between two domains of the polypeptide chain) ).
The Fc domains in the polypeptide chains of the DART™ molecules preferentially dimerize, resulting in the formation of a DART™ molecule that exhibits immunoglobulin-like properties, e.g., Fc-FcR interactions. Fc comprising diabodies may be dimers, for example comprised of two polypeptide chains each comprising a VH domain, a VL domain and an Fc domain. Dimerization of said polypeptide chains results in a bivalent DART™ comprising an Fc domain, albeit with a structure distinct from that of an unmodified bivalent antibody. Such DARTTM molecules will exhibit altered phenotypes relative to a wild-type immunoglobulin, e.g. altered serum half-life, binding properties, etc. In other embodiments, DART™ molecules comprising Fc domains can be tetramers. Such tetramers comprise two "heavier" polypeptide chains, i.e., a polypeptide chain comprising a VL, a VH and an Fc domain, and two "lighter" polypeptide chains, i.e., a polypeptide chain comprising a VL and a VH. The lighter and heavier chains interact to form a monomer, and said monomers interact through their unpaired Fc domains to form an Ig-like molecule. Such Ig-type DARTTM is tetravalent and can be monospecific, bispecific or tetraspecific.
[00264] The formation of a tetraspecific diabody molecule as described above requires the interaction of four different polypeptide chains. Such interactions are difficult to achieve efficiently within a single cell recombinant production system, due to many potential chain mismatches variables. One solution to increase the probability of mismatch is to design “buttons-in-holes” mutations in the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization. For example, with respect to Fc-Fc interactions, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a "button", eg tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will force the mutated domain to pair with a domain on which a complementary, or comfortable mutation has been designed, ie, “the hole” (eg, a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the diabody molecule, and further engineered into any portion of the polypeptide chains of said pair. Protein engineering methods to favor heterodimerization over homodimerization are well known in the art, in particular with respect to engineering immunoglobulin-like molecules, and are encompassed here (see, for example, Ridgway et al. (1996) ""Knobs -Into-Holes" Engineering Of Antibody CH3 Domain For Heacy Chain Heterodimerization," Protein Engr. 9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library," J. Mol. Biol.270:26-35, and Xie et al (2005) "A New Format Of Biespecific Antiboy: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J.Immunol.Methods 296:95-101; each of which is incorporated herein by reference in its entirety.
The invention also encompasses diabody molecules comprising variable Fc or articular variable Fc domains (or portion thereof), variable Fc domain comprising at least one amino acid modification (e.g. substitution, insertion or deletion) relative to an Fc domain comparable wild type or articular Fc domain (or portion thereof). Molecules comprising variable Fc domains or joint Fc domains (or portion thereof) (e.g. antibodies) typically have altered phenotypes relative to molecules comprising wild-type Fc domains or joint Fc domains or portions thereof. The variable phenotype can be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function as tested in a macrophage pendant or NK-dependent assay. The Fc domain modifications identified as the effector function of change are described above.
[00266] The present invention also encompasses molecules comprising a joint domain. The articular domain can be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM. In preferred embodiments, the articular domain is derived from IgG, wherein the IgG isotype is IgG1, IgG2, IgG3 or IgG4, or allotype thereof. Said articular domains can be designed onto a polypeptide chain comprising the diabody molecule with "an Fc domain such that the diabody molecule comprises a articular Fc domain. In certain embodiments, the Fc and articular domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments, the Fc and articular domain are separated by at least one other domain of the polypeptide chain, for example, the VL domain. The joint domains, or optionally the joint Fc domain, can be designed into a polypeptide of the invention at any position relative to other domains or portions of said polypeptide chain. In certain embodiments, a polypeptide chain of the invention comprises a hinge domain, which hinge domain is at the C-terminus of the polypeptide chain, wherein said polypeptide chain does not comprise an Fc domain. In still other embodiments, a polypeptide chain of the invention comprises a joint Fc domain, which joint Fc domain is at the C-terminus of the polypeptide chain. In other embodiments, a polypeptide chain of the invention comprises a joint Fc domain, which joint Fc domain is N-terminal of the polypeptide chain.
[00267] Each domain of the DARTTM polypeptide chain, that is, the VL, VH and Fc domain can be separated by a peptide linker. The peptide linker can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In certain embodiments, the amino acid linker sequence is GGGSGGGG (SEQ ID NO:52) encoded by the nucleic acid sequence ggaggcggat ccggaggcgg aggc (SEQ ID NO:53). The polypeptide chains of the DART™ molecule can be designed to comprise at least one cysteine residue that will interact with a counterpart cysteine residue in a second polypeptide chain of the DART™ to form an interchain disulfide bond. Such interchain disulfide bonds serve to stabilize the DARTTM molecule, thus improving expression and recovery in recombinant systems, resulting in a stable and consistent formulation, and improving the stability of the isolated and/or purified product in vivo. The cysteine residue can be introduced as a single amino acid or as part of a larger amino acid sequence, for example, a joint domain, into any portion of the polypeptide chain. In a specific embodiment, the cysteine residue can be designed to occur at the C-terminus of the polypeptide chain. In some embodiments, the cysteine residue is introduced into the polypeptide chain within the LGGC amino acid sequence. In a specific embodiment, the C-terminus of the polypeptide chains comprising the DARTTM molecule of the invention comprises the amino acid sequence LGGC (SEQ ID NO:54). In another embodiment, the cysteine residue is introduced into the polypeptide within an amino acid sequence comprising a articular domain, for example, EPKSCDKTHTCPP (SEQ ID NO:55) or ESKYGPPCPS (SEQ ID NO:56). In a specific embodiment, the C-terminus of a polypeptide chain of the DARTTM molecule of the invention comprises the amino acid sequence of an IgG articular domain, for example, SEQ ID No:55 or SEQ ID No:56. In another embodiment, the C-terminus of a polypeptide chain of a DARTTM molecule of the invention comprises the amino acid sequence VEPKSC (SEQ ID No:57), which can be encoded by the nucleotide sequence gttgagccca aatcttgt (SEQ ID No:58 ). In other embodiments, the cysteine residue is introduced into the polypeptide chain within the amino acid sequence LGGCFNRGEC (SEQ ID NO:59), which can be encoded by the nucleotide sequence ctgggaggct gcttcaacag gggagagtgt (SEQ ID NO:60). In a specific embodiment, the C-terminus of a polypeptide chain comprising the DARTTM of the invention comprises the amino acid sequence LGGCFNRGEC (SEQ ID NO:59). In still other embodiments, the cysteine residue is introduced into the polypeptide chain within the amino acid sequence FNRGEC (SEQ ID NO:61), which can be encoded by the nucleotide sequence ttcaacaggg gagagtgt (SEQ ID NO:62). In a specific embodiment, the C-terminus of a polypeptide chain comprising the DART™ of the invention comprises the amino acid sequence FNRGEC (SEQ ID NO:61).
[00268] In certain embodiments, the diabody molecule comprises at least two polypeptide chains, each comprising an amino acid sequence LGGC (SEQ ID NO:54) and are covalently linked by the disulfide bond between the cysteine residues in the sequences LGGC (SEQ ID NO:54). In another specific embodiment, the diabody molecule comprises at least two polypeptide chains, one comprising the sequence FNRGEC (SEQ ID NO:61) while the other comprises a articular domain (containing at least one cysteine residue), wherein the said at least two polypeptide chains are covalently linked by a disulfide bond between the cysteine residue in FNRGEC (SEQ ID NO:61) and a cysteine residue in the articular domain. In particular aspects, the cysteine residue responsible for a disulfide bond located in the articular domain is Cys-128 (as numbered according to Kabat EU; located in the articular domain of an intact, unmodified IgG heavy chain) and the residue of counterpart cysteine in SEQ ID NO:23 is Cys-214 (as numbered according to Kabat EU; located at the C-terminus of an intact, unmodified IgG light chain) (Elkabetz et al. (2005) “Cysteines In CHI Underlie Retention Of Unassembled Ig Heavy Chains” J. Biol. Chem. 280: 14402-14412). In still other embodiments, the at least one cysteine residue is designed to occur at the N-terminus of the amino acid chain. In still other embodiments, the at least one cysteine residue is designed to occur in the linker portion of the polypeptide chain of the diabody molecule. In still further embodiments, the VH or VL domain is designed to comprise at least one amino acid modification with respect to the parental VH or VL domain such that said amino acid modification comprises a replacement of a parental amino acid with cysteine.
[00269] In yet another aspect of that embodiment, domain (C) of the first polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57), derived from the articular domain of a human IgG, and which can be encoded by the sequence of nucleotide gttgagccca aatcttgt (SEQ ID NO:58). In another aspect of that embodiment, domain (F) of the second polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57). In certain aspects of that embodiment, domain (C) of the first polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO:61); and the (F) domain of the second polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57) or a joint domain. In other aspects of that embodiment, the (F) domain of the second polypeptide chain comprises the C-terminal 6 amino acids of the human kappa light chain, FNRGEC (SEQ ID NO:61); and domain (C) of the first polypeptide chain comprises the amino acid sequence VEPKSC (SEQ ID NO:57) or a joint domain.
As will be appreciated in light of the above, individual polypeptides of a bispecific DARTTM can form two species of homodimers and one species of heterodimer. In one embodiment of the present invention, a charged polypeptide can be added to the C-terminus of one, or more preferably, both DART™ polypeptides. By selecting charged polypeptides of opposite charge to the individual polypeptides of the bispecific DARTTM, the inclusion of such charged polypeptides favors the formation of heterodimers and decreases the formation of homodimers. Preferably, a positively charged polypeptide will contain a substantial content of arginine, glutamine, histidine and/or lysine (or mixtures of such amino acids) and a negatively charged polypeptide will contain a substantial content of aspartate or glutamate (or a mixture of such amino acids). Positively charged polypeptides that contain a substantial content of lysine and negatively charged polypeptides that contain a substantial content of glutamate are particularly preferred. In order to maximize the electrostatic attraction between such oppositely charged polypeptides, it is preferred to employ those polypeptides capable of spontaneously assuming a helical conformation.
[00271] Thus, in a preferred embodiment, a positively charged "E coil" will be attached to one of the polypeptides being used to form a bispecific DARTTM and a negatively charged "K coil" will be attached to the second of the DARTTM polypeptides. A particularly preferred E coil will have the sequence: (EVAALEK)4 [i.e. (SEQ ID NO:63) EVAALEKEVAALEKEVAALEKEVAALEK]. A particularly preferred K coil will have the sequence: (KVAALKE)4 [i.e. (SEQ ID NO:64) KVAALKEKVAALKEKVAALKEKVAALKE].
[00272] A preferred DARTTM polypeptide having such an E coil will have the general sequence: [VL Domain]—[GGGSGGGG]—[VH Domain]—[(EVAALEK)4]—GGGNS, where VL is the variable light Ig domain of DARTTMs, GGGSGGGG is SEQ ID NO:52, VH is the variable heavy LG domain of DARTTMs, (EVAALEK)4 is SEQ ID NO:63, and GGGNS is SEQ ID NO:65. A preferred DARTTM polypeptide having such a K-Coil will have the general sequence: [VL Domain]—[GGGSGGGG]—[VH Domain]—[(KVAALKE)4]—GGGNS, where VL is the variable light LG domain of DARTTMs, GGGSGGGG is SEQ ID NO:52, VH is the variable heavy LG domain of DARTTMs, (KVAALKE)4 is SEQ ID NO:64, and GGGNS is SEQ ID NO:65.
[00273] In yet one embodiment, the FHC regions can be connected to the E and/or K coils of the E-coil or K-coil DARTTMs. In addition, the separation between the Fc regions and the VH domain of DARTTM of an Fc-containing DARTTM it is desirable in cases where a less separate arrangement of such domains results in diminished interaction between such domains and their binding ligands or otherwise interferes with DART™ assembly. Although spacers of any amino acid sequence can be employed, it is desirable to employ spacers that form helical coils so as to maximally extend and project the Fc domain away from the variable domains. Because the oppositely charged folded polypeptides described above additionally function to promote heterodimeric formation, such molecules are particularly preferred spacers. Such bonin-containing Fc-DARTTM molecules provide benefits similar to those of Fc-DARTTMs, including improved serum half-life and recruitment of effector function. The E-coil and K-coil polypeptides described above are particularly preferred for this purpose. Thus, in a preferred embodiment, the DARTTM containing E-coil Fc will have the general sequence: [VL Domain]—[GGGSGGGG]—[VH Domain]—[(EVAALEK)4]—GGG—Fc Domain starting with D234 (Kabat numbering ), where VL is the variable light LG domain of DARTTMs, GGGSGGGG is SEQ ID No:52, VH is the variable heavy LG domain of DARTTMs, and (EVAALEK)4 is SEQ ID No:63. Similarly, in a preferred embodiment, the K-coil Fc containing DARTTM will have the general sequence: [VL domain]—[GGGSGGGG]—[VH domain]—[(KVAALKE)4]—GGG—Fc domain starting with D234 (Kabat numbering ), where VL is the variable LG domain of DARTTMs, GGGSGGGG is SEQ ID NO:51, VH is the variable heavy LG domain of DARTTMs, and (KVAALKE)4 is SEQ ID NO:64.
[00274] As indicated above, a coil-containing DARTTM molecule or a coil-containing Fc-containing DARTTM molecule may contain only one single coil separator, or it may contain more than one such separator (e.g., two separators, preferably from opposite charge, one of which is linked to each of the VH domain of DARTTM polypeptides). By binding the Fc region into such a spacer molecule(s), the ability to make bivalent, tetravalent, etc. versions. of Fc-DARTTM molecules by strand exchange is increased. Fc-DART™ molecules can thus be produced that form monomers or dimers depending on whether the Fc Domain is linked to one or both of the VH domains of DART™. 1. VERSATILITY OF DART™ B7-H3 MOLECULES
The bispecific DARTTMs of the invention can simultaneously bind two distinct and separate epitopes. In certain embodiments, the epitopes are from the same antigen. In other embodiments, the epitopes are from different antigens. In preferred embodiments, at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g., CD3, CD16, CD32, CD64, T cell receptor, etc.), which is expressed on lymphocytes T, natural killer (NK) cells or other mononuclear cells. In one embodiment, the DARTTM molecule binds to the effector cell determinant and also activates said effector cell. In this regard, the DART™ molecules of the invention may exhibit LG-like functionality irrespective of whether they still comprise an Fc domain (e.g., as tested in any effector function assay known in the art or exemplified herein (e.g., ADCC assay)). In certain embodiments, the bispecific DARTTM of the invention binds both a cancer antigen on a tumor cell and an effector cell determinant while activating said cell. In alternative embodiments, the bispecific DARTTM or DARTTM molecule of the invention can inhibit the activation of a target, e.g., effector, cell by simultaneously binding, and thus binding, an activating and inhibitory receptor on the same cell (e.g., binding both CD32A and CD32B, BCR and CD32B, or IgERI and CD32B) as described above (see, History Section). In yet one aspect of that embodiment, the bispecific DARTTM can exhibit antiviral properties by simultaneously binding two neutralizing epitopes on a virus (e.g., RSV epitopes; WNV epitopes such as E16 and E53). 2. DARTTM UNIVERSAL MOLECULES B7-H3
[00276] In one embodiment, the bispecific DARTTM molecules of the invention can be constructed to comprise an epitope binding domain that specifically binds to B7-H3 and a second epitope binding domain that specifically binds to a hapten, by example, fluorescein isothiocyanate (also known as fluoroisothiocyanate or FITC). Such DARTTM serves as a universal adapter ("UDARTTM"), capable of linking B7-H3 with molecules that interact with binding partners of fluorescein conjugates. For example, the FITC reactive arm of DART™ can be used to bind a FITC-labeled antibody that binds to a non-B7-H3 target involved in intercellular clustering, intercellular recruitment, cell-free recruitment, multiple targets, etc. The chimeric mouse Fc/Fc version of the anti-fluorescein MAb, 4420 can be employed as a source of FITC-specific CDR domains (Gruber, M. et al. (1994) “Efficient Tumor Cell Lysis Mediated By A Bispecific Single Chain Antibody Expressed In Escherichia coli,” J. Immunol. 152(11):5368-5374). 3. SPECIFIC TARGET CELL DART™ MOLECULES B7-H3
[00277] The bispecific DARTTM molecules of the invention offer unique opportunities to target specific cell types. For example, the bispecific DARTTM or DARTTM molecule can be designed to comprise a combination of epitope binding sites that recognize a set of antigens unique to a target cell or tissue type. Additionally, where one or both of the individual antigens is/are quite common separately in other tissue and/or cell types, low affinity binding domains can be used to construct the DARTTM or DARTTM molecule. Such low affinity binding domains will be unable to bind the individual epitope or antigen with sufficient avidity for therapeutic purposes. However, where both epitopes or antigens are present on a single target cell or tissue, the avidity of the DARTTM or DARTTM molecule for the cell or tissue, relative to a cell or tissue expressing only one of the antigens, will be increased accordingly. that said cell or tissue can be effectively targeted by the invention. Such a bispecific molecule may exhibit increased binding to one or both of its target antigens on cells expressing both said antigens with respect to a monospecific DARTTM or an antibody with a specificity for only one of the antigens.
[00278] For example, the B7-H3 specific DARTTMs of the present invention can be constructed to comprise a domain that is a binding ligand for a 2D Natural Terminator Group (NKG2D) receptor. The NKG2D receptor is expressed in human (and other mammalian) natural killer cells (Bauer, S. et al. (1999) "Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA," Science 285( 5428):727-729; Jamieson, AM et al (2002) “The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing,” Immunity 17(1):19-29) as well as on all CD8+ T cells (Groh, V. et al. (2001) "Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells" Nat. Immunol. 2(3):255-260; Jamieson, AM et al.( 2002) “The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing,” Immunity 17(1): 19-29). Such binding ligands, and particularly those that are not expressed in normal cells, include the histocompatibility molecule 60 (H60), the newly-inducible retinoic acid gene 1 (RAE-1) product, and the protein-like transcript 1 of murine UL16 binding (MULT1) (Raulet DH (2003) “Roles Of The NKG2D Immunoreceptor And Its Ligands,” Nature Rev. Immunol. 3:781-790; Coudert, JD et al. (2005) “Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells,” Blood 106: 1711-1717). Additional ligands reactive with human NKG2D include the MICA and MICB molecules related to the polymorphic class I MHC chain (Diefenbach, A. et al. (1999) "Natural Killer Cells: Stress Out, Turn On, Tune In," Curr. Biol 9(22):R851-R8533; Bauer, S. et al (1999) "Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA," Science 285(5428):727-729; Stephens, HA (2001) "MICA AND MICB Genes: Can The Enigma Of Their Polymorphism Be Resolved " Trends Immunol. 22:378-385. The MICA sequence is SEQ ID NO:66: MGLGPV FLLL AG I FPFAPPG AAAE P HS LRY LTVLSHDG N S L VQSGFLTEVH DÇQP LRC F and G QW RQKC JAK CF AE EV LG MKTWE 1'2! T TR DL GM U GK DLRMT AHIKDQKEGL HSLQEIRVCE IHEDNSTR5S QEFYYDGELF LSQNLETKEW TMPQSSRAQT LAMNVRNFLK EDAMKTKTHY HAMHADCLQE LRRYLKÊGW LRRTVPPMVM VTRSEASEGN ITVTCRASGF YPWNITLSWR QDGVSLSHDT QQWGDVLPDG NGTYQTWVAT RICQGEEQRF TCYMEHSGNH STHPVPSGKV LVLQSHWQTF HVSAVAAAAI FVIIIFYVRC CKKKTSAAEG PELVSLQVLD QHPVGTSDHR DATQLGFQPL MSDLGSTGST EGA
[00279] The MICB sequence is SEQ ID NO: 67: PHS LRYNLMV LS QDGSVQSG FLAE GH LDGQ P FL RY DRQK R RAKPQGQWAE DVLGAKTWDT ETEDLTENGQ DLRRTLTHIK DQKGGLHSLQ EIRVCEIHED SSTRGSRHFY YDGELFLSQH LETQESTVPQ 55RAQTLAMN VTKFWKEDAM KTKTHYRAMQ ADCLQKLQLP PMVWVICSEV SEGNITVTCR ASSFYPRNIT LTWRQD GV SLS H-J TQQWGDV LP DG NG1 TY QT W VATRIRQG E EQ RFTC YME H 5GNHGTHPVP SGKALVLOSO RTDFFYVSAA MPCFVTTTT7, CVFCCKKKTS AAEGP
Alternatively, the DARTTM molecules of the present invention can be constructed to comprise a domain that is a binding ligand for the T cell receptor ("TCR") or for CD3 (the T cell coreceptor). The TCR is naturally expressed by CD4+ or CD8+ T cells, and allows such cells to recognize antigenic peptides that are bound and presented by MHC class I or class II antigen presenting cell proteins. Recognition of a pMHC (peptide-MHC) complex by a TCR initiates the propagation of a cellular immune response that leads to cytokine production and lysis of the antigen presenting cell (see, for example, Armstrong, KM et al. ( 2008) “Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide-MHC Complexes,” Biochem. J. 415(Pt 2):183-196; Willemsen, R. (2008) “Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy,” Cytometry A. 73(11):1093-1099; Beier, KC et al. (2007) “Master Switches Of T-Cell Activation And Differentiation,” Eur. 29:804-812; Mallone, R. et al (2005) "Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes," Am. J. Ther. 12(6):534-550). CD3 is the TCR-binding receptor (Thomas, S. et al. (2010) "Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer," Immunology 129(2): 170-177; Guy, CS et al. (2009) “Organization Of Proximal Signal Initiation At The TCR: CD 3 Complex,” Immunol. Rev. 232(1):7-21 ; St. Clair, EW (Epub 2009 Oct 12) “Novel Targeted Therapies For Autoimmunity,” Curr. Opin. Immunol. 21(6):648-657; Baeuerle, PA et al. (Epub 2009 Jun 9) "Bispecific T-Cell Engaging Antibodies For Cancer Therapy," Cancer Res. ; Smith-Garvin, JE et al (2009) "T Cell Activation;" Annu. Rev. Immunol. 27:591-619; Renders, L. et al. (2003) "Engineered CD3 Antibodies For Immunosuppression" Clin. Exp Immunol. 133(3):307-309).
[00281] By constructing such DARTTM molecules to further comprise at least one epitope binding domain capable of binding to, for example, a receptor present on the surface of a target cell, such DARTTM molecules will be DARTTM molecules and so will be capable of binding to target cells and thereby causing the target cells to display the 2D Natural Terminator Group receptor binding ligand (NKG2D) or the TCR (whichever is present in the target cell binding DARTTM) (See, for example, Germain, C. et al. (2008) "Redirecting NK Cells Mediated Tumor Cell Lysis By A New Recombinant Bifunctional Protein", Prot. Engineer. Design Selection 21(11):665-672). Such DARTTMs can be used to redirect any desired target cell into a cell that is the target of NK cell-mediated cell lysis or T cell-mediated cytotoxicity. In one embodiment, the epitope binding domain of the DARTTM is capable of binding to a A receptor present on the surface of a target cell is an epitope that binds to a tumor associated antigen in order to redirect such cancer cells onto substrates for NK cell-mediated cell lysis or T cell-mediated cytotoxicity. Of particular interest are antigens. tumor associated which is a breast cancer antigen, an ovarian cancer antigen, a prostate cancer antigen, a cervical cancer antigen, a pancreatic carcinoma antigen, a lung cancer antigen, a bladder cancer antigen , a colon cancer antigen, a testis cancer antigen, a glioblastoma cancer antigen, an antigen associated with a B cell malignancy, an associated antigen with multiple myeloma, an antigen associated with non-Hodgkins lymphoma, or an antigen associated with chronic lymphocytic leukemia.
Suitable tumor associated antigens for such use include A33 (a colorectal carcinoma antigen; Almqvist, Y. 2006, Nucl Med Biol. Nov;33(8):991-998); B1 (Egloff, A.M. et al. 2006, Cancer Res. 66(1):6-9); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); beta-catenin ( Prange W. et al. 2003 J Pathol. 201(2):250-9); CA125 (Bast, R.C. Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81 ); CD5 (Calin, GA et al. 2006 Semin Oncol. 33(2):167-73; CD19 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD20 (Thomas, DA et al. 2006 Hematol Oncol Clin North Am. 20(5):1125-36); CD22 (Kreitman, RJ 2006 AAPS J. 18;8(3):E532-51); CD23 (Rosati, S. et al. 2005 Curr Top Microbiol Immunol. 5;294:91-107); CD25 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4): 139-48); CD27 (Bataille, R. 2006 Haematologica 91(9) ): 1234-40); CD28 (Bataille, R. 2006 Haematologica 91(9): 1234-40); CD36 (Ge, Y. 2005 Lab Hematol. ll(1):31-7); CD40/CD154 (Messmer , D. et al. 2005 Ann NY Acad Sci. 1062:51-60); CD45 (Jurcic, JG 2005 Curr Oncol Rep. 7(5):339-46); CD56 (Bataille, R. 2006 Haematologica 91(9) ): 1234-40); CD79a/CD79b (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48; Chu, PG et al. 2001 Appl Immunohistochem Mol Morphol. 9(2):97 -106); CD 103 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4): 139-48); CDK4 (Lee, YM et al. 2006 Cell Cycle 5(18):2110-4); CEA (antigen carcinoembryonic; Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46; Tellez-Avila, F.I. et al. 2005 Rev Invest Clin. 57(6):814-9); CTLA4 (Peggs, K.S. et al. 2006 Curr Opin Immunol. 18(2):206-13); EGF-R (epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer. 90 Spec No:S228-32); Erb (ErbB1; ErbB3; ErbB4; Zhou, H. et al. 2002 Oncogene 21(57):8732-40; Rimon, E. et al. 2004 Int J Oncol. 24(5): 1325-38); GAGE (GAGE-1; GAGE-2; Akcakanat, A. et al. 2006 Int J Cancer. 118(1):123-8); GD2/GD3/GM2 (Livingston, P.O. et al. 2005 Cancer Immunol Immunother. 54(10): 1018-25 ); gp100 ( Lotem, M. et al. 2006 J Immunother. 29(6):616-27 ); HER-2/neu ( Kumar, Pal S et al. 2006 Semin Oncol. 33(4):386-91 ); human papillomavirus-E6/human papillomavirus-E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; KSA (17-1A) (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80) ; MAGE (MAGE-1; MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); MART (Kounalakis, N. et al. 2005 Curr Oncol Rep. 7(5) :377-82; MUC-1 (Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46); MUM-1 (Castelli, C. et al. 2000 J Cell Physiol. 182(3) :323-31); N-acetylglucosaminyltransferase (Dennis, JW 1999 Biochim Biophys Acta. 6; 1473(1):21-34); p15 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7(9) :667-77); PSA (prostate-specific antigen; Cracco, CM. et al. 2005 Minerva Urol Nefrol. 57(4):301-11); PSMA (Ragupathi, G. 2005 Cancer Treat Res. 123:157- 80); sTn (Holmberg, LA 2001 Expert Opin Biol Ther. 1(5):881-91); TNF receptor (TNF-α receptor, TNF-β receptor; or TNF-Y receptor; van Horssen, R. et al. 2006 Oncologist. 11(4):397-408; Gardnerova, M. et al. 2000 Curr Drug Targets. 1(4):327-64); or VEGF receptor (O' Dwyer. P.J. 2006 Oncologist. 11(9):992-8).
[00283] Additional tumor associated antigens for such use (and publications describing antibodies specifically reactive to such antigens) include ADAM-9 (US Patent Publication No. 2006/0172350; PCT Publication No. WO 06/084075) ; ALCAM (PCT Publication No. WO 03/093443); Carboxypeptidase M (U.S. Patent Publication No. 2006/0166291); CD46 (U.S. Patent No. 7,148,038; PCT Publication No. WO 03/032814); Cytokeratin 8 (PCT Publication No. WO 03/024191); Ephrin receptors (and, in particular, EphA2 (US Patent No. 7,569,672; PCT Publication No. WO 06/084226); Integrin Alpha-V-Beta-6 (PCT Publication No. WO 03/087340 JAM-3 (PCT Publication No. WO 06/084078); KID3 (PCT Publication No. WO 05/028498); KID31 (PCT Publication No. WO 06/076584); LUCA-2 (Patent Publication US No. 2006/0172349; PCT Publication No. WO 06/083852); Oncostatin M (Oncostatin Receptor Beta) (US Patent No. 7,572,896; PCT Publication No. WO 06/084092); PIPA ( U.S. Patent No. 7,405,061; PCT Publication No. WO 04/043239); ROR1 (U.S. Patent No. 5,843,749); and Transferrin Receptor (U.S. Patent No. 7,572,895; Publication of PCT No. WO 05/121179).
[00284] Also of interest are antigens specific to particular infectious agents, e.g., viral agents including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza, human papilloma virus (HPV) ), foot and mouth (coxsackieviruses), rabies virus, herpes simplex virus (HSV), and causative agents of gastroenteritis, including rotavirus, adenovirus, calicivirus, astrovirus, and Norwalk virus; bacterial agents including, but not limited to, E. coli, Salmonella thyphimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Hemophilus influenzae, Shigella dysenteriae, Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumoniae .
[00285] In some embodiments, the molecules of the invention are designed to comprise an altered glycosylation pattern or an altered glycoform with respect to the comparable portion of the model molecule. Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, augmentation effector function. Engineered glycoforms can be generated by any method known to one skilled in the art, for example, using variant or engineered expression strains, by co-expression with one or more enzymes, for example, N-acetylglucosaminyltransferase III DI (GnTI11), expressing a DART™ of the invention in various organisms or cell lines from various organisms, or by modifying the carbohydrate(s) after the DART™ has been expressed and purified. Methods for generating engineered glycoforms are known in the art, and include, but are not limited to, those described in Umana et al. (1999) “Engineered Glycoforms Of An IgG1 Antineuroblastoma With Optimized Antibody-Dependent Cellular Cytotoxic Activity,” Nat. Biotechnol 17:176-180; Davies et al. (2001) “Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In Adcc Through Higher Affinity For Fc Gamma RIII,” Biotechnol Bioeng 74:288-294; Shields et al. (2002) "Lack Of Fucose On Human IgG1 N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity," J Biol Chem 277:26733-26740; Shinkawa et al. (2003) "The Absence Of Fucose But Not The Presence Of Galactose Or Bisecting N-Acetylglucosamine Of Human IgG1 Complex-Type Oligosaccharides Show The Critical Role Of Enhancing Antibody-Dependent Cellular Cytotoxicity," J Biol Chem 278:3466-3473) US 6,602 .684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAbTM glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See, for example, WO 00061739; EA01229125; US20030115614; Okazaki et al. (2004) "Fucose Depletion From Human IgG1 Oligosaccharide Enhances Binding Enthalpy And Association Rate Between IgG1 And FcGammaRIIIA," JMB, 336: 1239-49 each of which is incorporated herein by reference in its entirety.
[00286] The invention further encompasses incorporation of unnatural amino acids to generate the DARTTMs of the invention. Such methods are known to those skilled in the art such as those who use natural bis-synthetic machinery to allow the incorporation of all unnatural amino acids into proteins, see, for example, Wang et al. (2002) “Expanding The Genetic Code,” Chem. Comm. 1:1-11; Wang et al. (2001) "Expanding The Genetic Code Of Escherichia coli," Science, 292: 498-500; van Hest et al. (2001) “Protein-Based Materials, Toward A New Level Of Structural Control,” Chem. Comm. 19:1897-1904, each of which is incorporated herein by reference in its entirety. Alternative strategies focus on their enzymes responsible for amino acyl-tRNA biosynthesis, see, for example, Tang et al. (2001) "Biosynthesis Of A Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine In An Engineered Bacterial Host", J. Am. Chem. Soc. 123(44):11089-11090; Kiick et al. (2001) “Identification Of An Expanded Set Of Translationally Active Methionine Analogues In Escherichia coli,” FEBS Lett. 502(1-2):25-30; each of which is incorporated herein by reference in its entirety. In some embodiments, the invention encompasses methods of modifying a VL, VH or Fc domain of a molecule of the invention by adding or deleting a glycosylation site. Methods for modifying protein carbohydrate are well known in the art and encompass the invention, see, for example, U.S. Patent No. 6,218,149; EP 0 359 096 B1; US Publication No. US 2002/0028486; WO 03/035835; US Publication No. 2003/0115614; U.S. Patent No. 6,218,149; U.S. Patent No. 6,472,511; all of which are incorporated herein by reference in their entirety. VIII. METHODS OF USING B7-H3 MODULATORS AND ANTI-B7-H3 ANTIBODIES FOR THERAPEUTIC PURPOSES
[00287] Monoclonal antibodies to B7-H3 can be used for therapeutic purposes in individuals with cancer or other diseases. Anti-B7-H3 antibody therapy may involve both in vitro and in vivo complex formation as described above. In one embodiment, the anti-B7-H3 monoclonal antibody can bind to and reduce the proliferation of cancer cells. It is understood that the antibody is administered at a concentration that promotes binding under physiological conditions (eg, in vivo). In another embodiment, monoclonal antibodies to B7-H3 can be used for immunotherapy targeting cancer cells from different tissues like colon, lung, breast, prostate, ovary, pancreas, kidney and other cancers like sarcoma. In another embodiment, the anti-B7-H3 monoclonal antibody alone can bind and reduce cell division in the cancer cell. In another embodiment, anti-B7-H3 monoclonal antibody can bind cancer cells and delay metastasis development. In yet another embodiment, an individual with cancer receives palliative treatment with anti-B7-H3 antibody. Palliative treatment of an individual with cancer involves treating or lessening the adverse symptoms of the disease, or iatrogenic symptoms resulting from other treatments given for the disease without directly affecting the progress of the cancer. This includes treatments for pain relief, nutritional support, sexual problems, psychological distress, depression, fatigue, psychiatric disorders, nausea, vomiting, etc.
[00288] In such situations, the anti-B7-H3 antibody can be administered with agents that enhance or direct an individual's own immune response, such as an agent that strengthens ADCC...
[00289] In yet another embodiment, the anti-B7-H3 antibody can be conjugated or associated with a radioactive molecule, toxin (for example, calicheamicin), chemotherapeutic molecule, liposomes or other vehicles containing chemotherapeutic compounds and administered to an individual in need of such treatment to target these compounds to the cancer cell containing the antigen recognized by the antibody and thereby eliminate cancerous or diseased cells. Without being limited to any particular theory, the anti-B7-H3 antibody is internalized by the cell carrying B7-H3 on its surface, thereby delivering the conjugated moiety to the cell to induce the therapeutic effect. In yet another embodiment, the antibody can be employed as an adjunctive therapy at the time of surgical removal of a cancer expressing the antigen in order to delay metastasis development. The antibody can also be administered prior to surgery (neoadjuvant therapy) to an individual with a tumor expressing the antigen in order to shrink the tumor and thereby enable or simplify surgery, save tissue during surgery, and/or decrease the resulting disfigurement.
[00290] Dosing the cell cycle is considered in the practice of this invention. In such embodiments, a chemotherapeutic agent is used to synchronize the cell cycle of the tumor or other diseased target cells at a predetermined stage. Subsequently, administration of the anti-B7-H3 antibody of this invention (alone or in an additional therapeutic portion) is done. In alternative embodiments, an anti-B7-H3 antibody is used to synchronize the cell cycle and reduce cell division prior to administering a second phase of treatment; the second stage may be the administration of an anti-B7-H3 antibody and/or an additional therapeutic moiety.
Chemotherapeutic agents include radioactive molecules, toxins, also referred to as cytotoxins or cytotoxic agents, which includes any agent that is detrimental to the viability of cancer cells, agents and liposomes or other vesicles containing chemotherapeutic compounds. Examples of suitable chemotherapeutic agents include, but are not limited to, 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleucine, alkylating agents, sodium allopurinol, altretamine, amifostine, anastrozole, anthramycin (AMC)), antimitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, live BCG (intravesicular), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine, Colcicine, conjugated estrogens, Cyclophosphamide, Cyclotophamide, Cytarabine, Cytarabine, Cytoxanesin B, Dacarbazine, Dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCL, daunorubicin citrate, denileukin diftitox, D exrazoxane, Dibromomannitol, dihydroxyanthracindione, Docetaxel, dolasetrimesylate, doxorubicin HCL, dronabinol, E. coli L-asparaginase, emetin, epoetin alfa, Erwinia L-asparaginase, esterified estrogens, estradiol, estradiol, sodium ebrothin estramustine phosphate etidronate, citrororo factor etoposide, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoid, goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea, hydroxyurea interferon alfa-2b, irinotecan HCL, letrozole, calcium leucovorin, leuprolide acetate, levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL, mercaptipurine, methotrexamine, methotremycin , mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL, paclitaxel, p disodium amidronate, pentostatin, pilocarpine HCL, plimycin, polyfeprosan 20 (with carmustine implant), porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab, sargramostim, streptozotocin, tamoxifene, taxol, teniposide, teniposide chlorambucil, thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.
[00292] In a preferred embodiment, the cytotoxin is especially effective in dividing or rapidly dividing cells, such that non-dividing cells are relatively freed from toxic effects.
Antibodies of the invention can be internalized within the carcinoma cells or patients to which they bind and are, therefore, particularly useful for therapeutic applications, for example, distribution into the toxins of cells that need to be internalized by their adverse activities . Examples of such toxins include, but are not limited to, saporin, calicheamicin, auristatin, and maytansinoid.
Antibodies or polypeptides of the invention may be associated (including conjugated or linked) with a radioactive molecule, a toxin, or other therapeutic agents, or with liposomes or other vesicles containing therapeutic agents covalently or non-covalently, directly or indirectly. The antibody can be linked to a radioactive molecule, toxin, or chemotherapeutic molecule anywhere along the antibody so long as the antibody is capable of binding to its target B7-H3.
[00295] A toxin or chemotherapeutic agent may be administered concomitantly with (before, after, or during administration), or coupled (eg, covalently linked) to an appropriate monoclonal antibody directly or indirectly (eg, through a group linker, or, alternatively, through a binding molecule with appropriate coupling sites, such as a platform molecule as described in U.S. Patent No. 5,552,391). The toxin and chemotherapeutic agent of the present invention can be coupled directly to particular target proteins using methods known in the art. For example, a direct reaction between an agent and an antibody is possible when each has a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, such as one may be able to react with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (for example, one halide) in the other.
Antibodies or polypeptides can also be linked to a chemotherapeutic agent via a microcarrier. The term “microcarrier” refers to a biodegradable or non-biodegradable particle that is insoluble in water and that has a size of less than about 150 µm, 120 µm or 100 µm in size, more commonly less than about 50 to 60 µm, preferably less than about 10, 5, 2.5, 2 or 1.5 µm. Microcarriers include "nanocarriers", which are microcarriers having a size of less than about 1 µm, preferably less than about 500 nm. Such particles are known in the matter. Solid phase microcarriers can be particles formed from naturally occurring biocompatible polymers, synthetic polymers or synthetic copolymers, which can include or exclude microcarriers formed from agarose or crosslinked agarose, as well as other biodegradable materials known in the art. Solid phase biodegradable microcarriers can be formed from polymers that are degradable (eg, poly(lactic acid), poly(glycolic) acid and copolymers thereof) or that cause erosion (eg, poly(orthoesters) such as 3, 9-diethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane (DETOSU) or poly(anhydrides) such as sebacic acid poly(anhydrides) under physiological mammalian conditions. example, oil or lipid-based), such as liposomes, iscomes (immunity-stimulating complexes, which are stable complexes of cholesterol, and phospholipid, active adjuvant saponin) without antigen, or droplets or molecules found in oil-in-water or water emulsions in oil, provided with liquid phase microcarriers are biodegradable. Biodegradable liquid phase microcarriers typically incorporate a biodegradable oil, a number of which are known in the art, including squalene and vegetable oils. Microcarriers are typically spherical in shape, but microcarriers that deviate from spherical shape are also acceptable (eg, ellipsoid, wheel shape, etc.). Due to their insoluble nature (with respect to water), microcarriers are water-filterable and water-based (aqueous) solutions.
[00297] The conjugated antibody or polypeptide of the present invention may include a bifunctional linker that contains both a group capable of coupling with a toxic agent or chemotherapeutic agent and a group capable of coupling with an antibody. A linker can function as a spacer to distance an antibody from an agent to avoid interfering with binding capabilities. A linker can be cleavable or non-cleavable. A linker also serves to increase the chemical reactivity of a substituent or an agent or an antibody, and thereby increase coupling efficiency. An increase in chemical reactivity can also facilitate the use of agents, or functional groups in agents, that would not otherwise be possible. The bifunctional linker can be coupled to the antibody by means that are known in the art. For example, a linker containing an active ether moiety, such as an N-hydroxosuccinimide ester, can be used to couple to lysine residues in the antibody via an amide bond. In another example, a linker containing a nucleophilic amine or hydrazine residue can be coupled to aldehyde groups produced by the glycolytic oxidation of antibody carbohydrate residues. In addition to these direct coupling methods, the linker can be indirectly coupled to the antibody via an intermediate vehicle such as an aminodextran. In these embodiments the modified binder is via lysine, carbohydrate, or an intermediate vehicle. In one embodiment, the linker is site-selectively coupled to release thiol residues on the protein. Portions that are suitable for selective coupling to thiol groups on proteins are well known in the art. Examples include disulfide compounds, α-halocarbonyl and α-halocarboxyl compounds, and maleimides. When a nucleophilic amine function is present in the same molecule as a &-halo carbonyl or carboxyl group, there is the potential for cyclization to occur through intramolecular alkylation of the amine. Methods to prevent this problem are well known to the person of ordinary skill in the art, for example, by preparing molecules in which the amine and &-halo functions are separated by inflexible groups such as aryl or transalkene groups, which make undesirable cyclization stereochemically unfavorable. See, for example, U.S. Patent No. 6,441,163 for preparing conjugates of maytansinoids and antibody via a disulfide moiety.
[00298] One of the cleavable linkers that can be used for the preparation of antibody-drug conjugates is an acid-labile linker based on cis-aconitic acid that takes advantage of the acidic environment of different intracellular compartments like the endosomes found during mediated endocytosis per receptor and the liposomes. See, for example, Shen, W.C. et al. (1981) ("cis-Aconityl Spacer Between Daunomycin And Macromolecular Carriers: A Model Of pH-Sensitive Linkage Releasing Drug From A Lysosomotropic Conjugate" Biochem. Biophys. Res. Comtnun. 102:1048-1054 (1981)) for the preparation of conjugates of daunorubicin with macromolecular vehicles; Yang et al (1988) “Pharmacokinetics And Mechanism Of Action Of A Doxorubicin-Antibody Monoclonal 9.2.27 Conjugate Directed To A Human Melanoma Proteoglycan” J. Natl. Cane. Inst. 80:11541159) for the preparation of conjugates of daunorubicin to an anti-melanoma antibody; Dillman et al. (1988) (*Superiority Of An Acid-Labile Daunorubicin-Antibody Monoclonal Immunoconjugate Compared To Free Drug” Cancer Res. 48:6097-6102) to use an acid-labile ligand in a similar fashion to prepare conjugates of daunorubicin with an antibody of anti-T cell; and Trouet et al. (1982) “A Covalent Linkage Between Daunorubicin And Proteins That Is Stabel In Serum And Reversible By Lysosomal Hydrolases, As Required For A Lysosomotropic Drug-Carrier Conjugate: In Vitro And In Vivo Studies” Proc. Natl. Academic Sci. (U.S.A.) 79:626-629) to link daunorubicin to an antibody via a peptide spacer arm.
[00299] An antibody (or polypeptide) of this invention can be conjugated (bound) to a radioactive molecule or toxin by any method known in the art. For a discussion of methods for radiolabeled antibody (see, CANCER THERAPY WITH MONOCLONAL ANTIBODIES, D.M. Goldenberg (Ed.) CRC Press, Boca Raton, 1995). Suitable toxins include taxanes, maytansinoids, auristatins (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (such as those described in U.S. Patent Nos. 5,208,020; 5,416,064 ; 6,333,410; 6,340,701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497; 7,585,857; or 7,851,432), calicheamicin, anthracyclines (for example, doxorubicin), CC -1065 analogue, docetaxel,; cathepsin B or E; ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, and RNase; tiuxetan or toxic radioisotope (such as 90Y; 131I, 177Lu, 186Re, 188Re, 211At,212Bi,213Bi, 225Ac, etc.).
Alternatively, an antibody can be conjugated to a second antibody to form a heteroconjugate antibody as described in U.S. Patent No. 4,676,980. The formation of crosslinked antibodies can target the immune system to specific cell types, for example, diseased or cancer cells expressing B7-H3.
This invention also provides methods of delaying the development of metastases in an individual with cancer (including, but not limited to, prostate, lung, or kidney cancer) using an anti-B7-H3 antibody or other embodiments that bind to B7 -H3 in combination with a chemotherapeutic agent, or linked to a chemotherapeutic agent. In some embodiments, the antibody is a humanized or chimeric form of a non-human anti-B7-H3 antibody.
[00302] In yet another embodiment, the antibody can be employed as adjuvant therapy in the occasion of surgical removal of a cancer expressing the antigen in order to delay the development of metastases. The antibody or antibody associated with a chemotherapeutic agent may also be administered prior to surgery (neoadjuvant therapy) to an individual with a tumor expressing the antigen in order to shrink the tumor and thereby enable or simplify surgery, save tissue during surgery, and/or lessen the resulting disfigurement.
[00303] In yet another embodiment, any of the B7-H3 binding compositions described herein can bind to B7-H3-expressing cancer cells and induce an active immune response against cancer cells expressing B7-H3. In some cases, the active immune response can cause cancer cell death (eg, antibody binding to cancer cells inducing apoptotic cell death), or inhibit the growth (eg, block cell cycle progression) of cells. cancer cells. In other cases, any of the novel antibodies described herein can bind to cancer cells and antibody dependent cell cytotoxicity (ADCC) can eliminate cancer cells to which anti-B7-H3 binds. Therefore, the invention provides methods of stimulating an immune response comprising administering any of the compositions described herein.
[00304] In some cases, antibody binding may also activate both cellular and tumor responses and recruit more natural killer cells or increased cytokine production (eg IL-2, IFN-gamma, IL-12, TNF-alpha , TNF-beta, etc.) that further activate an individual's immune system to destroy cancer cells. In yet another embodiment, the anti-B7-H3s antibody can bind to cancer cells, and macrophages or other phagocytic cell can opsonize the cancer cells.
[00305] Various formulations of anti-B7-H3s antibodies or fragments thereof can be used for administration. In some embodiments, the anti-B7-H3s antibody or fragments thereof can be administered neat. In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that are well known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance or that facilitate the processing of the active compounds in preparations that can be used pharmaceutically for delivery to the site of action. For example, an excipient can give shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers.
Formulations suitable for parenteral administration include solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate for oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension can also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery to the cell.
[00307] The pharmaceutical formulation for systemic administration, according to the invention can be formulated for enteral, parenteral or topical administration. In fact, all three formulation types can be used simultaneously to perform systemic administration of the active ingredient. Excipients as well as formulations for parenteral and non-parenteral drug delivery are set out in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Edition, Lippincott Williams & Wilkins Publishing (2005). Formulations suitable for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof. Generally, these agents are formulated for administration by injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.), although other forms of administration (eg, oral, mucosal, etc.) may also be used. Therefore, anti-B7-H3 antibodies are preferably combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like.
The particular dosing regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history. Generally, a dose of at least about 100 µg/kg body weight, more preferably at least about 250 µg/kg body weight, even more preferably at least about 750 µg/kg body weight, even more preferably at less about 3 mg/kg body weight, even more preferably at least about 5 mg/kg body weight, even more preferably at least about 10 mg/kg body weight is administered.
[00309] Empirical considerations, such as the average lifespan, will generally contribute to determining the dosage. Antibodies, which are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the antibody's lifespan and to prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted during the course of therapy, and is based on reducing the number of cancer cells, maintaining the reduction of cancer cells, reducing the proliferation of cancer cells, or delaying the development of metastasis. Alternatively, sustained sustained release formulations of the anti-B7-H3s antibody may be appropriate. Various formulations and devices for carrying out sustained release are known in the art.
[00310] In one embodiment, dosages for anti-B7-H3 antibodies can be determined empirically in individuals who have received one or more administrations. Subjects receive increased dosages of an anti-B7-H3 antibody. To assess the effectiveness of the anti-B7-H3s antibody, a specific cancer disease state marker can be followed. These include direct measurements of tumor size through palpation or visual observation, indirect measurements of tumor size by X-ray or other imaging techniques; an enhancement as accessed by direct tumor biopsy and microscopic examination of the tumor specimen; the measurement of an indirect tumor marker (eg, PSA for prostate cancer), a decrease in pain or paralysis; improved speech, vision, breathing, and other impairments associated with the tumor; improved appetite; or an increase in quality of life as measured by accepted tests or prolonged survival. It will be apparent to the person skilled in the art that the dosage will vary depending on the individual, the type of cancer, the stage of the cancer, whether the cancer has begun to metastasize elsewhere in the individual, and the past and current treatments being used.
[00311] Other formulations include appropriate delivery forms known in the art including, but not limited to, vehicles such as liposomes. See, for example, Mahato et al. (1997) "Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives" Pharm. Res. 14:853-859. Liposomal preparations include, but are not limited to, cytofectins, multilamellar and unilamellar vesicles.
[00312] In some embodiments, more than one antibody may be present. Antibodies can be monoclonal or polyclonal. Such compositions can contain at least one, at least two, at least three, at least four, at least five different antibodies that are reactive against carcinomas, adenocarcinomas, sarcomas, or adenosarcomas. Anti-B7-H3 antibody can be mixed with one or more antibodies reactive against carcinomas, adenocarcinomas, sarcomas, or adenosarcomas in organs including, but not limited to, ovary, breast, lung, prostate, colon, kidney, skin, thyroid, bone, upper digestive tract, and pancreas. In one embodiment, a mixture of different anti-B7-H3s antibody is used. A mixture of antibodies, as they are often denoted in the art, can be particularly useful for treating a wide range of populations of individuals.
[00313] Having now generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified... EXAMPLE 1 IMMUNOISTOCOMPATABILITY INVESTIGATIONS
[00314] A panel of 49 mAbs was generated from tumor cell/fetal progenitor cell immunizations. Antibodies were evaluated for their ability to exhibit differential IHC dye tumor tissue relative to normal, non-cancerous tissue, ability to use in primate (and particularly cynomolgus monkey) antibody efficacy models, specificity affinity levels and levels of antigens with immunomodulatory activity and cellular internalization. 21 of the mAbs were initially identified by MS analysis and/or binding to B7-H3-CHO cells. The remaining 28 mAbs were identified by re-scanning the library by ELISA with B7-H3 protein. Characteristics of 46 of the 49 panel members are provided in Table 2.


[00315] IHC staining confirmed that the panel comprises antibodies that elicited a strong tumor for normal tissue binding differential in many of the identified antibodies, exhibited a range of binding properties by BIACORE™ analysis, exhibited reactivity for epitope variation overlapping and non-overlapping and exhibited a range of specificity for 4Ig vs. 2Ig B7-H3. The characteristics of the nine best candidates are shown in Table 3 and Table 4.



[00316] Table 5 provides a summary of the activity profiles of these antibodies.


An analysis of the activities of the antibodies shown in Table 6 revealed that their respective profiles differed and that each antibody was associated with both advantages and disadvantages relative to each other (Table 6).


[00318] Because BRCA84D, BRCA68D, BRCA69D and PRCA157 exhibit cleaner normal tissue IHC profiles, stronger, moderate to strong normal/tumor binding differential IHC (BIACORETM / IHC), cross-reactivity to B7-H3 from cynomologous monkeys, and potent UDARTTM activity, these antibody species were selected for further development. These antibodies differed from TES7 and OVCA2, which exhibited low affinity (in the BIACORETM assay), and non-cross-reactivity to B7-H3 from cynomolgus monkeys. These antibodies differed from SG27, which exhibited low affinity (in the BIACORE™ assay), lower IHC performance (weak binding) and lower UDART™ activity. These antibodies differed from GB8, which exhibited low affinity (in the BIACORETM assay), tumor-deficient normal/tumor IHC differential, and lower UDARTTM activity.
[00319] Using Caki-2 and Hs700T positive control cells, IHC investigations revealed that each of the antibodies exhibited a different optimal concentration and a different differential concentration relative to each other (Table 7).

Using the optimal and differential concentrations indicated in Table 7, the IHC responses of the B7-H3 antibodies in human tissues were determined. The results of these analyzes for Adrenal, Liver, Pancreatic, Kidney, Lung, and Colon are shown in Tables 8A-8B and Tables 9A-9B (all antibodies exhibited negative IHC responses to heart tissue).




Investigations conducted using cancer species showed that the B7-H3 antibodies of the present invention could be used to identify a diagnosis of cancer in multiple tissue sources (Table 10). In Table 10, the numbers indicate the number of plus signs (1 = +, 2 = ++, 3 = +++); each number referring to a different tested sample.


For prostate, breast, colon and lung cancer cells treated with the antibody B7-H3 BRCA84D, tumor sample dye was present in the tumor cells and stromal cells, including the tumor vasculature. In some tumor samples, stromal staining was stronger than tumor cells. When BRCA84D mAb was titrated to lower concentration, some cases showed reduced staining in tumor cells, but still retaining strong stromal stain. Upon staining with BRCA84D at 0.625 µg/ml, prostate cancer cells exhibited an IHC of 3/3+; breast cancer cells exhibited an IHC of 4/4+; colon cancer cells exhibited an IHC of 4/4+ and lung cancer cells exhibited an IHC of 4/4+. Upon staining with BRCA84D at 0.078 µg/ml, prostate cancer cells exhibited an IHC of 3/4+; breast cancer cells exhibited an IHC of 5/5+; colon cancer cells exhibited an IHC of 4/4+ and lung cancer cells exhibited an IHC of 3/4+.
Normal liver was treated with B7-H3 antibody BRCA68D, and staining was seen in hepatocytes and sinusoidal lining cells. Normal pancreas stained with B7-H3 antibody BRCA68D exhibited multifocal staining in collagen fiber and epithelium. Normal adrenal cells treated with B7-H3 antibody BRCA68D, exhibited staining in the cortex. Upon staining with BRCA68D at 0.156 μg/ml, gastric, renal and ovarian cancer cells all exhibited an IHC of 5/5+.
Additional IHC staining analyzes were conducted on tissue samples from gastric, kidney and ovarian cancer. The results of each analysis are shown in Table 12. In Table 11, the numbers indicate the number of plus signs (1= +, 2= ++, 3 = +++); each number referring to a different tested sample.

In summary, all mAbs tested showed varying degrees of staining intensity in normal liver, pancreas, colon and lung. Figure 1A shows the results of IHC investigations conducted using normal pancreas, liver, lung, and colon tissue specimens with BRCA84D at 0.625 μg/ml and 0.078 μg/ml. Liver staining was relatively restricted in sinusoidal lining cells (fibroblast and kupffer cells) with BRCA84D and TES7. OVCA22 showed membrane hepatocyte staining in addition to that of the sinusoidal lining cells at optimal concentration. However, staining in hepatocytes disappeared at the differential concentration. All other mAbs showed staining in hepatocytes including membrane or cytoplasm staining at both ideal and differential concentrations. Pancreas staining was observed in collagen fiber mainly and a small percentage of the epithelium (acinar cells or/and intercalated duct cells). Staining in the epithelium decreased or disappeared at differential concentration. Colon staining was relatively restricted in the apical membrane of the crypto epithelium and mucosal fibroblast. No binding was observed in colon lymphoid nodules. The lung showed very weak and uneven staining in the epithelium with BRCA84D, BRCA68D, BRCA69D and GB8. However, the color disappeared at the differential concentration. No staining was observed in the lung with TES7, OVCA22 and PRCA157 at both concentrations. Adrenal cortex staining was observed with almost all mAbs at optimal concentration, except BRCA84D. Adrenal staining obviously decreased with TES7 and OVCA22 at differential concentration. Heart and kidney staining did not show obvious staining with all mAbs (Figure IB). In view of these properties, BRCA84D was considered the best of the mAbs, followed in order by (2) TES7, (3) OVCA22, (4) the BRCA68D group, BRCA69D and PRCA157, and finally (5) GB8.
[00326] All mAbs included in the study showed positive staining in 4 types of cancer at optimal concentration. At differential concentration, BRCA84D still maintained good coloration in prostate cancer, breast cancer, and colon cancer. TES7 maintained good coloration in 4 types of cancer studies. The remaining mAbs showed various staining intensities in different tumor types. Tumor sample staining was observed in tumor cells and stromal cells, including vasculature. Some tumor samples showed positive staining only in the vasculature, ie, BRCA84D, BRCA69D, TES7, and PRCA157. Some tumors showed stronger stromal staining than tumor cell staining. When mAbs were titrated to the lowest concentration in these samples, some cases showed decreased or no staining in tumor cells but still maintained strong stromal staining. In general, in terms of expression in normal human tissues and differential expression in normal vs. of tumor, the mAb order from the best IHC performance to the worst performance is as follows: (1) BRCA84D, (2) TES7, (3) OVCA22, (4) the BRCA68D group, BRCA69D and PRCA157, and finally (5) GB8. Table 12 and Figure 2 show results for antibody BRCA84D.
EXAMPLE 2 B7-H3 CROSS REACTIVITY OF CYNOMOLOGIST MONKEY
[00327] The cynomolgus monkey B7-H3 sequence shares approximately 90% homology to its human counterpart, suggesting that the cynomolgus monkey is an excellent model for human B7-H3 interactions. Investigations were conducted to assess B7H3 cross-reactivity of BRCA84D, BRCA68D, BRCA69D, TES7, OVCA22 and PRCA157 candidates with adrenal, liver, kidney, pancreas and lung as well as a case of natural term placenta from cynomologist monkey, in order to compare any cross-reactivity with staining intensity and staining patterns observed for human tissues.
[00328] The staining concentration for each Mab tested is the optimal concentration that was determined in Caki-2 and Hs700T positive control cells (see, Table 8). Commercial goat anti-human B7-H3 (kino cross-reacted) was selected as a positive control antibody for stained cynomolgus placental tissue. Corresponding isotope controls were applied in each run of the experiments. The results of these investigations are shown in Table 13.


Investigation of IHC staining of BRCA84D (0.625 μg/ml) in cynomolgus placenta showed staining in deciduous cells but not in villi. No staining was observed in liver and cyno pancreas, however, sinusoidal cell staining was observed in human liver and localized fiber and epithelial staining was observed in human pancreas tissue.
Investigation IHC staining of BRCA68D (0.156 μg/ml) in cynomolgus placenta exhibited staining in deciduous cells, mesenchymal cells (endothelium and fibroblasts) and villi. Staining was present in hepatocyte membrane and liver fibroblast cytoplasm, as well as in pancreatic fiber and in the cytoplasm of pancreatic epithelium. Thus, human and cyne pancreatic and liver tissue exhibit similar staining patterns with BRCA68D.
In summary, BRCA84D, BRCA68D, BRCA69D and PRCA157 all showed cross-reactivity in kine tissues. BRCA84D showed no staining in monkey liver and pancreas; such staining has been observed in human pancreatic and liver tissues. BRCA68D and BRCA69D showed similar staining intensity and staining patterns in monkey tissues. Although BRCA68D, BRCA69D and PRCA157 showed staining pattern comparable to human tissues, staining intensity is not identical to human tissues under ideal conditions. TES7 and OVCA22 did not show any staining in monkey tissues under ideal conditions.
A summary of the comparative results of IHC staining in cynomolgus tissue and human tissue is provided in Table 14.

EXAMPLE 3 B7-H3 mAbs MEETS MULTIPLE ATCC CELL LINES
The antibodies of the present invention have been found to be capable of binding to multiple cancer cell lines contained in the collections of the American Type Culture Collection. Table 15 and Table 16 summarize the binding results.




EXAMPLE 4 B7-H3 MABS REDIRECTED EXTERMINATION
The antibodies of the present invention bind to B7-H3 present on the surface of cancer cells. Using conventional methods how antibodies can be labeled with fluorescein as described above. Using such labeled molecules are incubated in the presence of UDARTTM molecules having an epitope binding domain that binds to the T cell receptor and an epitope binding that binds to fluorescein ("TCR-UDARTTM"), they can bind to DARTTM and in this way they localize them to the surface of cells expressing B7-H3 and cause redirected killing. A. REDIRECT EXTERMINATION OF RENAL CARCINOMA CELLS A498
To demonstrate such redirected killing, fluorescein-labeled B7-H3 antibodies were incubated with such TCR-UDARTTM molecules and the ability of the molecules to mediate cytotoxicity of A498 renal carcinoma cells was assessed (Table 17). Based on the results achieved, the top candidates were concluded as being: RECA13, BRCA68D, BRCA69D and TDH6.


Renal carcinoma A498 cells were incubated with different concentrations of monoclonal antibodies reactive against B7-H3 in order to determine dose-dependent redirected antibody-mediated killing. The results of the experiments (Figures 3A-3B) showed that redirected killing was dose-dependent. B. REDIRECT EXTERMINATION OF A549 LUNG CANCER CELLS
To further demonstrate such redirected killing, fluorescein-labeled B7-H3 antibodies were incubated with the TCR-UDARTTM molecules described above or with UDARTTM molecules having an epitope binding domain that binds to CD16 and a binding domain of epitope that binds to fluorescein ("CD 16-UDARTTM"), and the ability of the molecules to mediate cytotoxicity of A549 lung cancer cells was evaluated (Table 18). The results of the experiments (Figures 3C-3D) showed that the redirected extermination was dose dependent. Based on the results achieved, the top candidates were concluded as being: BLA8, BRCA68D, BRCA69D and BRCA84D.

C. REDIRECT EXTERMINATION OF LNCAP PROSTATE CANCER CELLS
To further demonstrate such redirected killing, fluorescein-labeled B7-H3 antibodies were incubated with the TCR-UDART™ molecules described above or with UDART™ molecules having an epitope binding domain that binds to CD16 and a domain of epitope binding that binds to fluorescein ("CD 16-UDART™"), and the ability of the molecules to mediate cytotoxicity of LNcap prostate cancer cells was evaluated (Table 19). Based on the results achieved, the top candidates were concluded as being: BRCA68D, BRCA69D, BRCA84D and PRCA157.

EXAMPLE 5 CAPACITY OF MABS B7-H3 TO MEET WITH SOLUBLE B7H3-2IG AND SOLUBLE B7H3-4IG
[00339] As discussed above, B7-H3 exists in both a 4Ig domain-containing form (B7H3-4Ig) and a 2Ig domain-containing form (B7H3-2Ig). The anti-B7-H3 antibodies of the present invention were tested for their abilities to bind to soluble B7H3-2Ig (Figure 4A) and soluble B7H3-4Ig B7-H3 (Figure 4B). Antibodies have been found to exhibit a wide range of binding characteristics. Antibodies PRCA123, TDH5, BLA8, BRCA68D and SG24 were found to exhibit the strongest binding to B7H3-2Ig antibodies and antibodies TES7, LUCA50, BRCA165, OVCA22, ST09 and PA20 were found to exhibit the weakest binding to B7H3 -2Ig soluble. Antibodies PRCA123, BRCA69A, BLA8 and BRCA68D were found to exhibit the strongest binding to soluble B7H3-4Ig and antibodies TES7, OVCA21, BRCA 165 and ST09 were found to exhibit the weakest binding to soluble B7H3-4Ig. EXAMPLE 6 ANTIGEN AFINITY BINDING IN SOLUTION FOR CAPTURED MONOCLONAL ANTIBODIES
[00340] In order to demonstrate the binding affinity between antigens in solution and captured monoclonal antibodies, antibodies were captured Fc-specific Fab2 IgG fragments immobilized at a level of 100-200 RU. B7-H3 and B7-H3(4Ig) antigens were injected onto captured antibodies at a concentration of 100 nM (flow rate 20 µl/min for 120 sec, and binding was measured. Binding responses were normalized to the same level of binding). Captured mAb and the binding response for the m2B6 antibody control (mlgGl) was subtracted as empty.The results of this analysis (Figures 5A-5S; solid lines; B7-H3(4Ig) 100 nM; dotted lines; B7-H3, 100 nM)) demonstrated that the antibodies of the present invention exhibited strong binding to B7-H3(4Ig)... EXAMPLE 7 BIACORETM ANALYSIS: TITRATION OF B7-H3 MABS TO B7-H3 FIXED ASSETS
In order to demonstrate the relative binding affinities of B7-H3-2Ig and B7-H3-4Ig for the antibodies of the present invention, a BIACORETM analysis was performed. The B7-H3 antibodies of the present invention were allowed to bind to immobilized B7-H3-2Ig or to B7-H3-4Ig and the titration of binding over time was accessed (Figures 6A-6I). TDH5, PRCA123, BLA8, BRCA69 were found to have high affinity for both B7-H3-2Ig and B7-H3-4Ig. However, its epitope(s) have been found to be mostly barred on the B7-H3-4Ig molecule, with only a little being available. OVCA22 was found to have a very low affinity for both B7-H3-2Ig and B7-H3-4Ig with its epitope being equally available on both molecules. However, it is likely that only the B7-H3-4Ig form provides sufficient proximity for bivalent antibody binding (out of low rate), whereas B7-H3-2Ig can be bound only monovalently. TDH6 was found to have only any affinity in that format, with binding to 2Ig likely to be non-specific. TES7 and PA20 were found to be B7-H4-4Ig specific antibodies with low affinity. TES7 probably has a lower on rate and a higher off rate than PA20. BRCA84D was found to be an intermediate affinity antibody with a possibility of multiple binding sites on both B7-H3-2Ig and B7-H3-4Ig. Based on BIACORE™ analysis, BRCA84D due to its unusual binding site was considered a so-called antibody. TES7 and PA20 were considered candidates for specific binding to surfaces of high density antigens, and a high affinity-low specificity antibody (eg BRCA69D or other).
Figure 7 provides a comparison of BIACORE™ analysis of PRCA157, BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27 antibodies, illustrating that the anti-B7-H3s antibody of the present invention can exhibit a range of binding properties.
Figure 8 demonstrates the non-competitive specificity of several of the anti-B7-H3s antibodies of the present invention. In the experiment, human B7-H3 molecules were incubated in the presence of BRCA84D antibody and subjected to BIACORE™ analysis. After approximately 3 minutes a second anti-B7-H3 antibody was added to the reaction. If the second antibody competed with BRCA84D, it found the B7-H3 sites occluded and were unable to bind. The results indicate that antibodies BRCA68D, BRCA69D, and PRCA157 do not compete with BRCA84D for binding to human B7-H3. EXAMPLE 8 ANTI-B7-H3 mAbs INTERNALIZE IN CSC AND ATCC CELL LINES
The ability of the anti-B7-H3s antibody of the present invention to become internalized upon binding to cancer cells was investigated. Prostate CSC cells and Hs700t pancreatic cells were incubated with an anti-B7-H3 antibody. Cell viability was determined after incubation in the presence of a saporin-conjugated secondary anti-mouse antibody that will be toxic to the cells if bound to the primary antibody and internalized. The results of this investigation for prostate CSC cells (Figure 9A) and for pancreatic Hs700t cells (Figure 9B) demonstrate the ability of the antibodies of the present invention to become internalized into cells. EXAMPLE 9 B7-H3 mAb BINDING AND ELISA CROSS-BLOCK ANALYSIS
In order to explore the cross-reactivity of the antibodies of the present invention and the epitopes recognized by such antibodies, the extent of binding occurring in the presence of a competing antibody B7-H3 was measured. The results of this analysis are shown in Figures 10A-10F, and show that BRCA68D competes with BRCA69D. TES7 and OVCA22 were also found to compete with each other, but TES7 and not OVCA22 were also found to compete with both BRCA68D and BRCA69D. GB8 was found to compete with SG27 for binding to B7-H3-2Ig but not to B7-H3-4Ig. The data are summarized in Table 20 and show at least four distinct epitopes for B7-H3-4Ig (ie, the epitope recognized by SG27, the epitope recognized by GB8, the epitope recognized by OVCA22 and TES7, and the epitope recognized by BRCA68D , BRCA69D and TES7) and at least two epitopes for B7-H3-2Ig (i.e. the epitope recognized by SG27 and GB8, and the epitope recognized by BRCA68D and BRCA69D).


[00346] The attributes of the key anti-B7-H3s antibody of the present invention are shown in Table 21. Based on its displayed differential staining of normal and cancer tissues, its abilities to bind B7-H3-4Ig as well as B7-H3 -2Ig, its binding affinities are measured by the BIACORE™ analysis described above and its abilities to bind to cynologues B7H3, antibodies, BRCA68D, BRCA69D, BRCA84D, and PRCA157 were judged to be the most preferred antibodies.

EXAMPLE 10 HUMANIZED ANTIBODY ANTI-B7-H3S
The monoclonal antibody BRCA84D has been humanized to produce antibodies (generally referred to herein as "hBRCA84D") offering enhanced human therapeutic potential. The sequences of the variable light chain, and the variable heavy chain, and their respective amino acid and polynucleotide sequences of the resulting humanized antibody (designated herein as "hBRCA84D-1") are provided below:
[00348] Humanized BRCA84D-1 Variable Light Chain (SEQ ID NO:68): DI QLTQS P 3 E L S A3V GDRVT 1TCK ASQNVD TNVAWY QQK P GKAPKLLIY S ASYRYSGVPS EFSGSGSGTD FTLTISSLQP EDFATYYCTFKQQ YNK
[00349] Polynucleotides Sequences Encoding Light Chain Variable BRCA84D-1 Humanized (SEQ ID NO: 69): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgacc atcacatgca aggcctccca gaacgtggac accaacgtgg cctggtatca gcagaagcct ggcaaggccc ctaagctgct gatctactcc gcctcctacc ggtactccgg cgtgccttcc aggttctccg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggacttcg ccacctacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc tggaaatcaa g
[00350] Variable Light Chain BRCA84D-1 Humanized CDRi (SEQ ID NO:70):

Polynucleotide Sequence Encoding Variable Light Chain BRCA84D-1 CDRi (SEQ ID NO:71): aaggccagtc agaatgtgga tactaatgta gcc
[00352] Variable Light Chain BRCA84D-1 Humanized CDR2 (SEQ ID NO:72):

Polynucleotide Sequence Encoding Variable Light Chain BRCA84D-1 Humanized CDR2 (SEQ ID NO:73): tcggcatcct accggtacag t
[00354] Variable Light Chain BRCA84D-1 Humanized CDR3 (SEQ ID NO:74):

Polynucleotide Sequence Encoding Variable Light Chain BRCA84D-1 Humanized CDR3 (SEQ ID NO:75):

[00356] Humanized BRCA84D-1 Variable Heavy Chain Amino Acid Sequence (SEQ ID NO:80): EVQLVESGGG LVQFGGSLRL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY IS S DS S S A1Y YA DT VK GR E 'TI SR DM AK t J DT AV YYC ARGR ENIYYGSRLD YWGQGTTVTV SS
[00357] Polynucleotides Encoding Sequences Variable Heavy Chain Humanized BRCA84D-1 (SEQ ID NO: 81): gaggtgeagc tggtcgagtc tggcggagga ctggtgcagc ctggcggctc cctgagactg tcttgcgccg cctccggctt caccttctcc agcttcggca tgcactgggt ccgccaggct ccaggcaagg gactggaatg ggtggcctac atctcctccg actcctccgc catctactac gccgacaccg tgaagggcag gttcàôcatc túccgggaca acgccaagaa ctccótgtàí; etgeagatga actccôtgeg ggacgaggae accgccgtgt act act gogó eagaggecgg gagaatatct aetacggctc ccggctggat tattggggcc agggcaccac cgtgaccgtg LccLcL
[00358] Variable Heavy Chain BRCA84D-1 Humanized CDR1 (SEQ ID NO:82):

Polynucleotide Sequence Encoding Variable Heavy Chain BRCA84D-1 Humanized CDR1 (SEQ ID NO:83):

[00360] Variable Heavy Chain BRCA84D-1 Humanized CDR2 (SEQ ID NO:84):

Polynucleotide Sequence Encoding Variable Heavy Chain BRCA84D-1 Humanized CDR2 (SEQ ID 85): tacattagta gtgacagtag tgccatctac tatgcagaca cagtgaag
[00362] Variable Heavy Chain BRCA84D-1 Humanized CDR3 (SEQ ID NO:86):

Polynucleotide Sequence Encoding Variable Heavy Chain BRCA84D-1 Humanized CDR3 (SEQ ID NO:87):

Figures 11A-11B show the alignment of variable light chain (Figure 11A) or variable heavy chain (Figure 11B) amino acid residues of BRCA84D and its humanized derivative, hBRCA84D...
In order to obtain hBRCA84D species that exhibit improved affinity for human B7-H3, polynucleotides encoding hBRCA84D-1 light or heavy chains (i.e., hBRCA84D-1VL or hBRCA84D-1VH, respectively) were subjected to mutagenesis and hBRCA84D-1 swapped light chain derivatives hBRCA84D-2VL, hBRCA84D-3VL, hBRCA84D-4VL, hBRCA84D-5VL, and hBRCA84D-6VL and swapped hBRCA84D-1 heavy chain derivatives hBRCA84D-2VH, hBRCA84D-3VH, and hHBRCA84V have been isolated and characterized. The amino acid and polynucleotide sequences of the variable light and heavy chains of these antibodies are shown below:
[00366] hBRCA84D-2VL (SEQ ID NO:89): DIQLTQSPSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YNNYPFTFGQ GTKLEIK
[00367] Polynucleotide Encoding hBRCA84D-2VL (SEQ ID NO: 90): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgacc atcacatgca aggcctccca gaacgtggac accaacgtgg cctggtatca gcagaagcct ggcaaggccc ctaaggcgct gatctactcc gcctectacc ggtactcegg egtgccttee aggttctceg getαcggetc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggaetteg ccacctaeta ctgccagcag tacaacaact accctttcac ctteggccag ggcaccaagc g tggaaatcaa
[00368] hBRCA84D-3VL (SEQ ID NO:91): DIQLTQSPSF LSASVGDRVS VTCKASQNVD TNVAWYQQKP GKAPKLLIYS AS YRYSGVPS RF■ SGSGSGTD FTLTISSLQP E DFATYYCQQ Y N N Y P E’TFKQ
[00369] Polynucleotide Encoding hBRCA84D-3VL (SEQ ID NO: 92): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgtcc gtcacatgca aggcctccca gaacgtggac accaacgtgg cetggtatca gcagaagcct ggcaaggcce etaagetget gatetactce gcctcctacc ggtactccgg cgtgccttcc aggtteteeg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggaetteg ceacctacta ctgccagcag tacaaeaact accctttcac cttcggccag ggcaccaagc g tggaaatcaa
[00370] hBRCA84D-4VL (SEQ ID NO:93): DIQLTQSPSF LSASVCDRVT ITCKASQNVD TNVAWYQQKP GQAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDEATYYCQQ YNNYPFTFGQ GTKLEIK
[00371] Polynucleotide Encoding hBRCA84D-4VL (SEQ ID NO: 94): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgac eatcac atgca aggcc ac tc ca ACE gaaegtggac the cgtgg cctggtatca geagaagcct ggccaggccc etaagetget gateLaci.cc gcctcctacc ggtactccgg cgtgccttcc aggtteteeg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggaetteg ceacctacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc tggaaatcaa g
[00372] hBRCA84D-5VL (SEQ ID NO:95): DIQLTQSFSF LSASVGDRVT ITCKASONVD TNVAWYQQKF GQAPKALIYS AS ¥ RY S GV P S R F' S G S G S GT D FT LTIS S LQP E DE AT ¥ Y CQQK Y N Y N GT
[00373] Polynucleotide Encoding hBRCA84D-5VL (SEQ ID NO: 96) gacatccagc tgacαcagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgacc atcacatgca aggcctccca gaacgtggac accaacgtgg ectggtatca gcagaagcct ggccaggccc ctaaggcgct gatctactcc gcctcctacc ggtactccgg cgtgccttcc aggttctccg gctccggctctggcaccgac ttcaccctga ccatctccag cctgcagcct gaggacttcg ccacctacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc g tggaaatcaa
[00374] hBRCA84D-6VL (SEQ ID NO:97): DIQLTQSFSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKLLIYS ASYRYSGVPS RFSGSGSGTD FTLTISSLQP EDFAEYYCQQ YNNYFFTFGQ GTKLEIK
[00375] Polynucleotide Encoding hBRCA84D-6VL (SEQ ID NO: 98) gacatccagc tgacαcagtc cccctcct tc ctgtctgcat ccgtgggcga cagagtgacc atcacatgca aggcctccca gaacgtggac accaacgtgg cctggtatca gcagaagcct ggcaaggccc ctaagctgct gatctactcc gcctcctacc ggtactccgg cgtgccttcc aggttctccg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggacttcg ccgagtacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc tggaaatcaa g
[00376] hBRCA84D-2VH (SEQ ID NO:99): EVQLVESGGG LVQPGGSLFL SCAASGFTFS SFGMHWVRQA PGKGLEWVAY 1S S DS S AIY Y A DT VK GR F'T1 S R DN AKNS LY LQMN S LRQ TV D T AV YWG
[00377] Polynucleotide Encoding hBRCA84D-2VH (SEQ ID NO: 100): gaggtgcagc tggtcgagtc tggcggagga ctggtgcagc ctggcggctc cctgagactg tcttgcgccg cctccggctt caccttctcc agcttcggca tgcactgggt ccgccaggct ccaggcaagg gactggaatg ggtggcctac atctcctccg actcctccgc catctactac gccgacaccg tgaagggcag gttcaccatc tcccgggaca acgccaagaa ctccctgtac ctgcagatga actccctgcg ggacgaggac accgccgtgt actactgcgg cagaggccgg gagaatatct actacggctc ccggctggat tattggggcc agggcaccac eg t ga c egt gt ec tet
[00378] hBRCA84D-3VH (SEQ ID NO:101): EVQL VE S GGG L VQ P G G S LRL S C AASG ∑'T ES S F'GMHW VRQ A PGKG LE WVAY ISSDSSAIYY ADTVKGRFTI SRDNAKNSLY LQMNSSQGRTJ YWGTV YWG
[00379] Polynucleotide Encoding hBRCA84D-3VH (SEQ ID NO: 102): gaggtgcagc tggtcgagtc tggcggagga ctggtgcagc ctggcggctccctgagactg tcttgcgccg cctccggctt caccttctcc agcttcggcatgcactgggt ccgccaggct ccaggcaagg gactggaatg ggtggcctacatctcctccg actcctccgc catctactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtac ctgcagatgaactccctgcg ggacgaggac accgccatgt actactgcgg cagaggccgggagaatatct actacggctc ccggctggat tattggggcc agggcaccaccgtgaccgtg tcctct
[00380] hBRCA84D-4VH (SEQ ID NO:103): AND VQI. VP, 5 GGG LVQ PG G S LR L SC AA S G PT RS S FGMf 1WVRQ A PG KG LE WV A Y ISSDSSA1YY ADTVKGRE'TI SRDNAKNSLY LQMNSENSED TAVYYCARGR ENIYYGSRLD YWGQGTTVTV SS
[00381] Polynucleotide Encoding hBRCA84D-4VH (SEQ ID Nθ: 104): gaggtgcagc tggtcgagtc tggcggagga ctggtgcagc ctggcggctccctgagactg tcttgcgccg cctccggctt caccttctcc agcttcggcatgcactgggt ccgccaggct ccaggcaagg gactggaatg ggtggcctacatctcctccg actcctccgc catαtactac gccgacaccg tgaagggcaggttcaccatc tcccgggaca acgccaagaa ctccctgtac ctgcagatgaactccctgcg gagcgaggac accgccgtgt actactgcgc cagaggccgggagaatatct actacggctc ccggctggat tattggggcc agggcaccaccgtgaccgtg tcctct
[00382] Table 22 lists the variable light chain hBRCA84D and variable heavy chain mutations studied, the numbers refer to the Kabat numbering system used in Figures 11A and 11B.


The relative binding affinities of light chain derivatives hBRCA84D, hBRCA84D-3VL, hBRCA84D-4VL and hBRCA84D-5VL to human B7-H3 were determined by the formation of antibodies containing these light chain variable regions and a heavy chain BRCA84D-1VH chimeric (Figure 12). BRCA84D-5VL (K42Q, L46A) was found to have the highest binding affinity of the hBRCA84D-VL tested. BRCA84D-5VL was thus used as the light chain to investigate the relative binding affinities of hBRCA84D heavy chains of hBRCA84D-1VH, hBRCA84D-2VH, hBRCA84D-3VH and hBRCA84D-4VH for human B7-H3 (Figure 13). hBRCA84D-2VH (A93G) was found to have the highest binding affinity of the tested hBRCA84D-VH.
[00384] The amino acid and polynucleotide encoding sequences of chimeric BRCA84D-1 are as follows:
[00385] chBRCA84D Light Chain (SEQ ID NO: 105): DTAMTQSOKF MSTSVGDRVS VTCKASQMVD TNVAWYQQKP GQSPKALIYS ASYRYSGVFD RFTGSGSGTD FTLTINNVQS EDLAEYFCQQ YNNYPFTFGS GTKLEIKRTV AAPSVFIFEE SDEQLKSGTA SWCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC
[00386] Polynucleotide Encoding Light Chain chBRCA84D (SEQ ID NO: 106): gacattgcga tgacccagtc tcaaaaattc atgtccacat cagtaggaga, cagggtcagc gtcacctgca aggccagtca gaatgtggat actaatgtag cctggtatca acagaaacca gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaacaa tgtgcagtct gaagacttgg cagagtattt ctgtcagcaa tataacaact atccattcac gttcggctcg gggacaaagt tggaaataaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gttag
[00387] Heavy Chain chBRCA84D (SEQ ID NO:107):L VOL VE S GGG LVQ PG GS RKL SC AASG FT t'S S FGMHWVROA PE KG LE WVAY IS SDSSA1YY ADTVKGRE"I11 SRDNP.:<NTL,F LQMTS TAM. YCGRGR ENIY YGS RAD YWGQGTTLT SSASTKGP £ V VF PLAP $$ KST SGGTAAL L CL VK VT VS DY FPE F HNS GA T, TS GV FTP PAVLQ SSG I. YS TS SV VTV PSSSL GT QTYICFIVNHK PSNTKVDKFV EPKSCDKTHT CPPCPAPELL GGPSVELFPP KPKDTLMISR TPEVTCWVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRWSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKLSLSP GA
[00388] Polynucleotide Encoding the Heavy Chain chBRCA84D (SEQ ID NO: 108): gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct ccagagaagg ggctggagtg ggtcgcatac attagtagtg acagtagtgc catctactat gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgg aagagggagg gaaaacattt actacggtag taggcttgac tactggggcc aaggcaccac tct caca gt. ct cc tcagcctccac ca the GG GCCC until GGT and 11 cc cc ctgg caccctcctc caagagcacc tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagagagtt gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ÇÇtggtggac gtgagccacg aagacectga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct
The relative binding affinities of antibodies containing: (1) hBRCA84D-2VL and hBRCA84D-2VH (two tests), (2) BRCA84D chimeric, (3) antibody containing hBRCA84D-5VL and BRCA84D-HC chimeric, and (4 ) antibody containing hBRCA84D-5VL and hBRCA84D-2VH were compared. The results are shown in Figure 14. EXAMPLE 11 HUMANIZED ANTI-B7-H3S ANTIBODIES INHIBIT TUMOR GROWTH IN XENOGRAFTS
In order to demonstrate the ability of humanized anti-B7-H3 antibodies to inhibit tumor growth in vivo, tumor growth of HT-1197 urinary bladder carcinoma cells and A498 renal carcinoma cells was studied in a murine xenograft. The humanized hBRCA84D-2 antibody (VL chain hBRCA84D-2 / VL chain hBRCA84D-2) was modified to comprise an Fc region having L235V, F243L, R292P, Y300L, and P396L substitutions. The modified Fc-antibody hBRCA84D-2 was administered to mice (at a dose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) 7 days, 14 days, 21 days, and 28 days after cell implantation cancerous. The results show that at all doses the Fc-modified hBRCA84D-2 antibody administered was able to inhibit tumor growth of HT-1197 urinary bladder carcinoma cells (Figure 15) and A498 renal carcinoma cells (Figure 16) . EXAMPLE 12 B7-H3 RECEIVER AND T-CELL-SPECIFIC DUAL-Affinity Redirection Reagents (DartTMs) Mediate Powerful Redirected T-Cell Termination
Dual affinity redirection reagents (DARTTMs) specific for B7-H3 and the T-cell receptor ("TCR") and for the 2D Natural Terminator Cell Receptor Group (NKG2D) were prepared. Such DARTTMs have the ability to locate a T cell (by binding such a T cell to the TCR binding portion of a TCR binding DARTTM) or locate an NK cell (by binding such an NK cell to the TCR binding portion NKG2D of an NKG2D binding DART™ to the site of a cancer cell (by binding such cancer cell to the B7-H3- binding portion of the DART™). The localized T cell or NK cell can then mediate the cancer cell terminator in a process termed here the "redirected" terminator.
The dual affinity redirection reagent (DARTTM) specific for B7-H3 and the T cell receptor ("TCR") was considered to have the hBRCA84D-2 anti-B7-H3 variable domains and anti- TCR:
[00393] TCR coil DARTTM chain VL x hBRCA84D VH-2-E (SEQ ID NO:109): ETVLTQSFAT LSLSPGERAT LSCSATSSVS YMHWYQQKPG KAPKRWIYDT SKLASGVFSR FSGSGSGTEF TLTISSLQPE DFATYYCQSFAT LSCSATSSVS YMHWYQQKPG 'GMHW VRQAPGKGLE WVAYISSDSS AIYYADTVKG RFTISRDNAK NSLYLQMWSL RDE.DTAVYYC GRGRENIYYG SRLDYWGQGT TVTVSSGGCG GGEVAALEKE VAALEKZVAA LEKEVAALEK
[00394] Polynucleotide Encoding TCR VL x coil Chain hBRCA84D VH-2e DART ™ (SEQ ID NO: 110): gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccaggggaaagagccacc ctctcctgca gtgccacctc aagtgtaagt tacatgcactggtatcagca gaaaccaggg aaagccccta agcgctggat ctatgacacatccaaactgg cttctggggt c ^ catcaagg ttcagcggca gtggatctgggacagaattt actctcacaa tcagcagcct gcagcctgaa gattttgcaacttattactg tcagcagtgg agtagtaacc cgctcacgtt tggccaggggaccaagcttg AQA "caaagg aggcggatcc ggcggcggag gcgaqgLqcagctggtcgag tctggcggag gactggtgca gcctggcggc tccctgagactgtcttgcgc cgcctccggc ttcaccttct ccagcttcgg catgcactgggtccgccagg ctccaggcaa gggactggaa tgggtggcct acatctcctccgactcctcc gccatctact acgccgacac cgtgaagggc aggttcaαcatcL cccgg g caacgccaa g AACT ccctgtacc tg ca gatgaac TCCC t.gcgggacgagg acaccgccgt gtactactgc ggcagaggcc gggagaatatctactacggc tcccggctgg attattgggg ccagggcacc accgtgaccgtqtcctccqg aggat-gtggc gqtggagaag tcgccgcact ggag^a^xgagStt^ctcjctt t^gagaagga ct-tja-aaagr^cctcpgagaaa
[00395] hBRCA84DVL coil Chain VH-TCR x 2 -K (SEQ ID NO: 111): DIQLTQSFSF LSASVGDRVT ITCKASQNVD TNVAWYQQKP GKAPKALIYS ASYRYSGVPS RPSGSGSGTD FTLTISSLQP EDEATYYCQQ YMNYPPTFGQ CTKLEIKOGfi SGGGGQVQLV QSGAEVKKPG ASVKVSCKAS GYKFTSYVMH WVRQAPGQGL EwIGYIN PY DVT N G K YNEK FK RVTITA DKS T STAY LQ MNS LRSEDTAVHY CARGSYYDYD GEVYWGQGTL VTVSSgQCGC ÇKVAALKEKV AALKEKVAAL KEKVAAT.KE
[00396] Polynucleotide Encoding hBRCA84DVL-2 x coil chain TCR V H - K (SEQ ID NO: 112): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgacc atcacatgca aggcctccca gaacgtggac accaacgtgg cctggtatca gcagaagcct ggcaaggccc ctaaggcgct gatctactcc gcctcctacc ggtactccgg cgtgccttcc aggttctccg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggacttcg ccacctacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc tggaaatcaa gggaggcgga tccggcggcg gaggecaggt tcagctggtg cagtctggag ctgaggtgaa gaagcctggg gcctcagtga aggtctcctg caaggccagc ggttacaagt ttaccagcta cgtgatgcac tgggtgcgac aggcccctgg acaagggctt gagtggatcg gatatattaa tccttacaat gatgttacta agtacaatga gaagttcaaa ggcagagtca cgattaccge ggacaaatce acgagcacag cctacctgca gatgaacagc ctgagatccg aggacacggc cgtgcactac tgtgcgagag ggagctacta tgattacgac gggtttgttt actggggcca agggactctg gtcactgtga gctccggagg atgtggcggt gqaaaagtgg ccscactgaa gg ^ aaacj-.t Sctcjαtttcja aac_agaagg_t cgççgcactt aac^aaaagg tcgcacjccct
A dual-affinity redirection reagent (DARTTM) specific for B7-H3 and the Natural Terminator 2D Group receptor (NKG2D) was considered to have the hBRCA84D-2 anti-B7-H3 variable domains and anti- TCR:
[00398] DARTTM Chain of Coil NKG2D VL x hBRCA84D VH-2-E (SEQ ID NO: 113) : QSALIQ PASV S GS P GQ SITI SCSGSSSMIG NMA VN WY QQ L PGKAPKLL1Y YDDLLPsGVs DRFSGSKSGT AFGTS GS GS GSPV GM H WVRQ A PG KGLE WV AY T 5 S D5 5 AIYY AD T VK GE FT IS R DN AK l-151. Y 7 .Q MN S LRDEDTA V Y YCGGRREM IY Y G SR LDYW G QG TIVT V SS GGCGGGEVAA LEKEVAALEKE VAALEKEVA ALEK
[00399] Polynucleotide Encoding DARTTM chain NKG2D coil VL x hBRCA84D VH-2-E (SEQ ID NO: 114): cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc aatcaccatc tcctgttctg gaagcagctc caacatcgga aataatgctg ttaactggta ccagcagctc ccaggaaagg ctcccaaact cctcatctat tatgatgacc tactgccctc aggggtctct gaccgattct ctggctccaa gtctggcacc tcagccttcc tggαcatcag tgggctccag tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtcriagtg ttcggcggag ggaccaagct gaccgtccta ggaggcggat ccggcggcgg aggcgaqgLq cagctggtcg agtαtggcgg aggactggtg cagcctggcg gctccctgag actgtcttgc gccgcctccg gcttcacctt ctccagcttc ggcatgcact gggtccgcca ggctccaggc aagggactgg aatgggtggc ctacatctcc tccgactcct ccgccatcta ctacgccgac accgtgaagg gcaggttcac catctcccgg gacaacgcca agaactccct gtacctgcag atgaactccc tgcgggacga ggacaccgcc gtgtactact gcggcagagg ccgggagaat atctactacg gctcccggct ggattattgg ggccagggca ccaccgtgac cgtgtcctcc ggaggatgtg gcggtggaga ac∑£cjccgca ctggagaaag ag^ttgctgc tttggacjaag cacttjaaaa Sc_g:ritggaiga. yy
[00400] Coil chain hBRCA84DVL-2 x NKG2D VH - K (SEQ ID NO: 115):

[00401] Polynucleotide Encoding coil Chain hBRCA84DVL-2 x NKG2D VH-K (SEQ ID NO: 116): gacatccagc tgacccagtc cccctccttc ctgtctgcct ccgtgggcga cagagtgacc atc acatgca aggcctccca gaacgtggac ac ac to cctggtatca gcagaagcct cgtgg ggcaaggccc ctaaggcgct gatctactcc gcctcctacc ggtactccgg cgtgccttcc aggtteteeg gctccggctc tggcaccgac ttcaccctga ccatctccag cctgcagcct gaggaetteg ccacctacta ctgccagcag tacaacaact accctttcac cttcggccag ggcaccaagc tggaaatcaa gggaggcgga tccggcggcg gaggccaggt acagctggtg gagtctgggg gaggcctggt caagcctgga gggtccctga gactctcctg tgcagcgtct ggattcacct tcagtagcta tggcatgcac tgggtccgcc aggctccagg caaggggctg gagtgggtgg catttatacg gtatgatgga agtaataaat actatgeaga ctccgtgaag ggccgattca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc ctgagagctg aggacacggc tgtgtattac tgtgcgaaag atcgaggttt gggggatgga acctactttg aetactgggg ecaagggacc acggtcaccg cctcctccgg aggatgtggc ggtgga: iag tcgccgcact ^aaggagaaa gttgctgctt tgaaagagaa ggtcgccgca cttaaggaaa aggtcgcagc 2£tc|aaacjací
[00402] In order to demonstrate the ability of DARTTMs to mediate such redirected killing of cancer cells, the above-described hBRCA84D-2 / anti-TCR DART™ ("T-DART™"), hBRCA84D-2, hBRCA84D-2 (Fc -modified: L235V, F243L, R292P, Y300L, and P396L), and a TCR-DARTTM control were incubated at various concentrations with target cancer cells (SK-MES-1 lung cancer cells, A498 renal carcinoma cells, LNCaP cells prostate cancer cells, or UACC-62 melanoma cells) and PBMC remnant effector (E:T ratio = 30:1) and cytotoxicity was determined (LDH Assay). The results of these investigations are shown in Figures 17A-17D and demonstrate the ability of hBRCA84D-2 / anti-TCR DART™ ("T-DARTTM") to mediate targeted killing of cancer cells. EXAMPLE 13 PHARMACOKINETIC PROFILE IN MICE WITHOUT TUMOR
The anti-B7-H3 antibody (Mab1) was injected into male mice mCD16-/-, hCD16A_FOXN1 (5 mg/kg; IV) and serum was assayed (pre-dose e) at 2, 15, 30 min, and 1, 2, 4, 6 hours, and 1, 2, 3, 6, 8, 14, 21, and 28 days after injection. The antibody was found to have a T ^ of 10.54 days and one of 43.493 µg/ml. Antibody concentration over time was found to be biphasic, fitting into a two-component model (Figures 18A-18B). Predicted pharmacokinetic profiles generated using a 2-compartment model with 5 mg/kg dose parameters are shown in Figure 18C. EXAMPLE 14 ABILITY OF ANTI-B7-H3 ANTIBODY TO BAND TO CANCEROUS CELLS OF URINARY BLADDER HT-1197 AND PREVENT OR INHIBIT TUMOR DEVELOPMENT IN A MURINE XENOGRAFT MODEL
The anti-B7-H3 antibody (Mab1) described above was accessed for its ability to bind HT-1197, a urinary bladder carcinoma line expressing human B7-H3. As shown in Figure 19, such cells exhibit greater expression of PRCA135 than HER2, and thus are particularly suitable for accessing the therapeutic potential of the antibodies of the present invention to remediate HT-1197 tumors. In accordance with this finding, anti-B7-H3 antibody variants hBRCA84D were found to be capable of binding to HT-1197 cells. Figure 20 shows binding affinity of Mab1 antibodies for HT-1197 cells.
[00405] Mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 8 x 106 HT-1197 cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab 1 treatment was initiated within 7 days of implantation via iv Q7D x5 using doses of 0.1, 0.5, 1, 5, or 10 mg/kg (eight female mice per dose). Centuximab (anti-EGRF antibody) was administered to a control group of mice at doses of 1, 5, or 15 mg/kg (eight female mice per dose). Eight female mice were also injected with vehicle or with a control of 10 mg/kg IgG. Tumor measurements were taken every 3-4 days. The results of the experiment (Figure 21A) show that Mab 1 was able to prevent or inhibit the development of urinary bladder tumor in a murine graft model. Figure 21B shows the results obtained using centimab. A comparison of Figures 21A and 21B demonstrates that the antibodies of the present invention are more effective than centimab in preventing or inhibiting urinary bladder tumor development in the murine xenograft model. Figure 21C compares the results obtained at the maximum doses tested. EXAMPLE 15 ABILITY OF ANTI-B7-H3 ANTIBODY TO BAND TO CANCEROUS CELLS OF URINARY BLADDER HT-1376 AND PREVENT OR INHIBIT TUMOR DEVELOPMENT IN A MURINE XENOGRAFT MODEL
The anti-B7-H3 antibody (Mab1) described above was accessed for its ability to bind HT-1376, a human B7-H3- urinary bladder carcinoma cell line. As shown in Figures 22A-22B, such cells exhibit greater expression of PRCA135 than HER2 or PMSA, and thus are particularly suited to access the therapeutic potential of the antibodies of the present invention in remediating HT-1376 tumors. In accordance with this conclusion, the anti-B7-H3 hBRCA84D antibody variables were found to be capable of binding to HT-1197 cells. Figures 22A-22B show the binding affinity of Mab1 antibodies for HT-1197 cells.
[00407] Mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 5 x 106 HT-1376 cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab 1 treatment was started within 7 days of implantation via iv Q7D x4 at a dose of 1 mg/kg. The results of the experiment (Figure 23) show that Mab1 was able to prevent or inhibit urinary bladder tumor development in the murine xenograft model. EXAMPLE 16 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO BAND TO CANCER CELLS
[00408] Anti-B7-H3 antibody BRCA84D was accessed through FACS analysis for its ability to bind: colorectal cancer cells SW480 and SW620; gastric cancer cells AGS; M-14 cell melanoma and LOX IMVI; 22rv prostate cancer cells; and pancreatic cancer cells AsPC-1 and BxPc-3; and A498 and 786-0 renal cancer cells. The antibody was found to be able to bind to all such cells. EXAMPLE 17 ABILITY OF ANTI-B7-H3 ANTIBODY TO PREVENT OR INHIBIT GASTRIC TUMOR DEVELOPMENT IN A MURINE XENOGRAFT MODEL
[00409] The mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 5 x 106 AGS cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was initiated within 7 days of implantation via iv Q7D x5 using doses of 0.5, 1, 5, or 10 mg/kg. The results of the experiment (Figure 24) showed that Mab1 was able to prevent or inhibit gastric tumor development in a murine xenograft model. EXAMPLE 18 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO BAND TO CANCEROUS LUNG CELLS AND PREVENT OR INHIBIT TUMOR DEVELOPMENT IN A MURINE XENOGRAFT MODEL
A549 lung cancer cells were incubated in the presence of hBRCA84D, chBRCA84D and hBRCA84 (0264 Fc) variables and the cytotoxic effect of these antibodies was determined. The results of this experiment are shown in Figure 25, and indicate that all three of the antibodies were cytotoxic to A549 cells.
[00411] The mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 8 x 106 A549 cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was initiated within 7 days of implantation via iv Q7D x4 using a dose of 1 mg/kg. The results of the experiment (Figure 26) show that Mab1 was able to prevent or inhibit the development of cancerous lung tumor in the murine xenograft model.
[00412] FACS analysis was conducted on CaLu3 lung cancer cells in order to determine whether such cells bind anti-B7-H3s antibody. The experiment confirmed that such cells express B7-H3 and bind to the antibodies of the present invention. To determine whether the antibodies of the present invention were effective in preventing or inhibiting lung cancer tumor development, mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 5 x 106 CaLu3 cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was initiated within 7 days of implantation via iv Q7D x4 using a dose of 0.5, 1, or 5 mg/kg. The results of the experiment (Figure 27) show that Mab1 was able to prevent or inhibit lung cancer tumor development in the murine xenograft model. EXAMPLE 19 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO PREVENT OR INHIBIT THE DEVELOPMENT OF A LOX MELANOMA TUMOR IN A MURINO XENOGRAFT MODEL
[00413] The mice (eight female mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with LOX-IMVI melanoma cancer cells and then inoculated with iv/Q7D x3 with PBS control, IgG control (5/ mg/kg), Mab1 (0.5, 1, 5 or 10 mg/kg), or ip/BIWx2 with Doxetaxel (5, 10 or 20 mg/kg). Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was started within 7 days of implantation. The results of the experiment (Figures 28A-28C) show that Mab1 was able to prevent or inhibit melanoma cancer tumor development in a murine xenograft model. EXAMPLE 20 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO PREVENT OR INHIBIT THE DEVELOPMENT OF UACC-62 MELANOMA TUMOR IN A MURINO XENOGRAFT MODEL
[00414] The mice (eight female mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with UACC-62 melanoma cancer cells and then inoculated iv/Q7D x5 with PBS control, IgG control (5/mg/kg ) or Mab1 (0.5, 1, 5 or 10 mg/kg). Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was started within 7 days of implantation. The results of the experiment (Figure 29) show that Mab1 was able to prevent or inhibit prostate cancer tumor development in the murine xenograft model. EXAMPLE 21 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO PREVENT OR INHIBIT 22RV PROSTATE TUMOR DEVELOPMENT IN A MURINO XENOGRAFT MODEL
[00415] The mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 6 x 106 22rv of prostate cancer cells and then inoculated iv/Q7D x4 with control PBS, IgG (10 mg/kg), Mab1 (0.5, 1, 5, or 10 mg/kg; Q7D x5) or Trastuzumab (1.7 or 15 mg/kg). Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was started within 7 days of implantation. The results of the experiment (Figures 30A-30C) show that Mab1 was able to prevent or inhibit prostate cancer tumor development in the murine xenograft model. EXAMPLE 22 ABILITY OF THE ANTI-B7-H3 ANTIBODY TO BAND TO KIDNEY CANCER CELLS AND PREVENT OR INHIBIT TUMOR DEVELOPMENT IN A MURINE XENOGRAFT MODEL
A498 renal cancer cells were incubated in the presence of the variants hBRCA84D, chBRCA84D and hBRCA84 (0264 Fc) and the cytotoxic effect of these antibodies was determined. The results of this experiment are shown in Figure 31, and indicate that all three of the antibodies were cytotoxic to A498 cells.
[00417] A498 renal cancer cells were incubated in the presence of the variants hBRCA84D, chBRCA84D and hBRCA84 (0264 Fc) and the cytotoxic effect of these antibodies was determined. The results of this experiment are shown in Figure 31, and indicate that all three of the antibodies were cytotoxic to A498 cells.
IHC analysis of A498 graft tumor tissue was conducted using biotinylated BRCA84D antibody (20 µg/ml), BRCA69D (5 µg/ml) and anti-Her2 antibody (20 µg/ml). The BRCA84D antibody was found to bind in 20 to 40% of tumor tissue (weekly or moderately: + or ++); BRCA69D was found to bind in 80 to 100% of tumor tissue (moderately to strongly: ++ or +++). The BRCA84D antibody was found to bind weakly in 40% of the UMUC-3 (+) tumor tissue; BRCA69D was found to moderately or strongly bind in 70% of each tumor tissue (++ or +++); it was found that anti-Her2 antibody variably binds in 20% of such tumor tissue (+ -+++). As controls, anti-Her2 antibody was found to bind to SKBR-3 cells (+++) and BRCA84D and BRCA69D were found to be able to bind to Hs 700T cells (+++)
[00419] The mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 5 x 106 A498 renal cancer cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGEL™. Mab1 treatment was initiated within 7 days of implantation via iv Q7D x5 using doses of 0.1, 0.5, 1, 5, or 10 mg/kg. Centuximab (anti-EGRF antibody) was administered to a control group of mice at doses of 1, 7, or 15 mg/kg. Additional control mice were injected with vehicle or with 10 mg/kg of IgG control. The results of the experiment (Figure 32) show that Mab1 was able to prevent or inhibit kidney cancer tumor development in the murine xenograft model.
[00420] The mice (mCD16-/-, hCD16A+_FoxN1) were alternatively implanted subcutaneously in their flanks with 5 x 106 786-0 renal cancer cells. Tumor cells were implanted in 200 µl of Ham's F12 Medium diluted 1:1 with MATRIGELTM. Mab1 treatment was initiated within 7 days of implantation via iv Q7D x5 using doses of 0.1, 0.5, 1, 5, or 10 mg/kg. Centuximab (anti-EGRF antibody) was administered to a control group of mice at doses of 1, 7, or 15 mg/kg. Additional control mice were injected with vehicle or with 10 mg/kg of IgG control. The results of the experiment (Figures 33A-33B) show that Mab1 was able to prevent or inhibit kidney cancer tumor development in the murine xenograft model.
The activity of Mab1 was compared with that of paclitaxel, a mitotic inhibitor used in cancer chemotherapy. Groups of eight female mice (mCD16-/-, hCD16A+_FoxN1) were implanted subcutaneously in their flanks with 786-0 renal cancer cells and then provided with Mab1 via iv Q7D at doses of 0.1, 0.5, 1, 5, or 10 mg/kg. Paclitaxel was administered to a control group of eight such mice at a dose of 2.5 mg/kg on days 21, 28, and 35 of the study. Additional control mice (seven females per group) were injected with vehicle or with 5 mg/kg of IgG control. The results of the experiment (Figure 34) show that Mab 1 was able to prevent or inhibit kidney cancer tumor development in the murine xenograft model. EXAMPLE 23 TOXICOLOGY STUDY OF CYNOMOLOGICAL MONKEYS
[00422] A toxicology study of cynomolgus monkeys is conducted in order to assess acute toxicology profile after a single dose of Mab1, determine the pharmacokinetic profile for Mab1, establish a time vs. relationship. dose for induction of cytokines associated with effector cell activation, and to assess the effect of drug treatment at the level of circulating leukocytes (eg, NK and T-cells).
[00423] Such a study can be designed to involve four groups of 6 monkeys (3 males and 3 females) and to extend 7 weeks from initial treatment to final necropsy. Group 1 would comprise a control group that would receive vehicle only for weeks 1 and 2. Four members of Group 1 (two males and two females) would be euthanized at week 3. The remaining members of Group 1 would receive additional vehicle at week 3 and would be sacrificed for necropsy at week 7. Groups 2-4 are experimental groups that would receive vehicle at week 1, and antibody B7-H3 (1, 30, or 100 mg/kg, respectively) at week 2. Four members of each group ( two males and two females) would be sacrificed at week 3. The remaining members of each Group would receive additional vehicle at week 3 and would be sacrificed for necropsy at week 7.
[00424] All infusions are well tolerated and no mortality or significant changes in body weight, clinical signs or serum chemistry are observed. Dose-dependent reductions in circulating NK cells but not circulating B and T- cells are observed.
[00425] The study provides verification of cynomologist monkey as a relevant toxicological species. When contacted with normal human tissue, the BRCA84D antibody showed varying degrees of staining intensity in the liver, pancreas, colon, lung and adrenal cortex. Liver staining was relatively restricted to sinusoidal lining cells (fibroblast and kupffer cells). Pancreas staining was observed mainly in the collagen fiber and a small percentage of the epithelium (acinar cells and/or intercalated duct cells). Colon staining was relatively restricted in the apical membrane of the crypto epithelium and mucosal fibroblast. The lung showed very weak and uneven coloration in the epithelium. BRCA84D showed good cross-reactivity in cynomologist monkey tissues compared to human tissue profile with the exception of lack of staining in liver and pancreas, and possible expression of B7-H3 in cynomologist monkey pituitary cells.
[00426] All publications and patents mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference in their entirety. Although the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification and this application is intended to cover any variations, uses, or adaptations of the invention generally following the principles of the invention and including such departures from the present description as come within known customary practice within the art to which the invention belongs and as may be applied to the essential features set forth hereinafter.

权利要求:
Claims (24)
[0001]
1. ISOLATED ANTIBODY OR ITS IMMUNOREATIVE FRAGMENT, characterized in that said isolated antibody or said fragment comprises a variable domain that specifically binds to an extracellular domain of B7-H3, wherein said antibody or said fragment thereof comprises: (A ) a light chain variable domain comprising CDR1 (SEQ ID NO: 21), CDR2 (SEQ ID NO: 23) and CDR3 (SEQ ID NO: 25), and a heavy chain variable domain comprising CDR1 (SEQ ID NO: :29), CDR2 (SEQ ID NO:31) and CDR3 (SEQ ID NO:33); or (B) a light chain variable domain comprising CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9) and a heavy chain variable domain comprising CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17); or (C) a light chain variable domain comprising CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 39) and CDR3 (SEQ ID NO: 41) and a heavy chain variable domain comprising CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 49).
[0002]
The ISOLATED ANTIBODY according to claim 1, characterized in that said antibody or fragment thereof comprises said light chain variable domain comprising CDR1 (SEQ ID NO:21), CDR2 (SEQ ID NO:23) and CDR3 (SEQ ID NO: 25), and said heavy chain variable domain comprising CDR1 (SEQ ID NO: 29), CDR2 (SEQ ID NO: 31) and CDR3 (SEQ ID NO: 33).
[0003]
The ISOLATED ANTIBODY according to claim 1, characterized in that said antibody or fragment thereof comprises said light chain variable domain comprising CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9), and said heavy chain variable domain comprising CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17).
[0004]
The ISOLATED ANTIBODY according to claim 1, characterized in that said antibody or fragment thereof comprises said light chain variable domain comprising CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 39) and CDR3 (SEQ ID NO: 41), and said heavy chain variable domain comprising CDR1 (SEQ ID NO: 45), CDR2 (SEQ ID NO: 47) and CDR3 (SEQ ID NO: 49).
[0005]
The ISOLATED ANTIBODY according to any one of claims 1 to 4, characterized in that it binds to B7-H3 which is expressed endogenously on the surface of a cancer cell.
[0006]
The ISOLATED ANTIBODY according to any one of claims 1 to 5, characterized in that said antibody or its fragment is internalized after binding to said B7-H3 expressed on the surface of said cancer cell.
[0007]
The ISOLATED ANTIBODY of any one of claims 1 to 6, characterized in that said antibody comprises a human IgG1 Fc variant region, wherein said human IgG1 Fc variant region comprises at least one related amino acid modification(s) to a wild-type Fc region, said amino acid modification(s) comprising amino acid modification(s) which alter(s) the affinity or avidity of said variant Fc region to bind to an FCYR of so that said antibody exhibits increased effector function relative to said wild-type Fc region.
[0008]
8. THE ISOLATED ANTIBODY according to claim 7, characterized in that said modification of the Fc region comprises: (A) at least one substitution of the group consisting of: (1) F243L;(2) D270E;(3) R292P;(4 ) S298N; (5) Y300L; (6) V305I; (7) A330V; and (8) P396L; (B) at least one substitution of two amino acid residues, said substitutions being selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; and(3) R292P and V305I;(C) at least one substitution of three amino acid residues, said substitutions being selected from the group consisting of: (1) F243L, R292P and Y300L;(2) F243L, R292P and V305I;(3 ) F243L, R292P and P396L; and (4) R292P, V305I and P396L; (D) at least one substitution of four amino acid residues, said substitutions being selected from the group consisting of: (1) F243L, R292P, Y300L and P396L; e(2) F243L, R292P, V305I and P396L; or (E) a substitution of at least five amino acid residues: F243L, R292P, Y300L, V305I and P396L; wherein said numbering is in accordance with the Kabat numbering scheme.
[0009]
The ISOLATED ANTIBODY of claim 8, wherein said antibody comprises the substitutions of: (A) F243L, R292P, and Y300L; (B) L235V, F243L, R292P, Y300L, and P396L; or (C) F243L, R292P, Y300L, V305I, and P396L; wherein said numbering is in accordance with the Kabat numbering scheme.
[0010]
The ISOLATED ANTIBODY according to claim 9, characterized in that said antibody comprises substitutions of: L235V, F243L, R292P, Y300L and P396L, wherein said numbering is in accordance with the Kabat numbering scheme.
[0011]
11. ISOLATED ANTIBODY according to claim 9, characterized in that said antibody comprises: (A) a light chain variable domain comprising CDR1 (SEQ ID NO: 5), CDR2 (SEQ ID NO: 7) and CDR3 (SEQ ID NO: 9) and a heavy chain variable domain comprising CDR1 (SEQ ID NO: 13), CDR2 (SEQ ID NO: 15) and CDR3 (SEQ ID NO: 17); and (B) a modification of the Fc region comprising the substitutions: L235V, F243L, R292P, Y300L, and P396L; wherein the numbering is in accordance with the Kabat numbering scheme.
[0012]
The ISOLATED ANTIBODY according to any one of claims 1 to 11, characterized in that said antibody is a chimeric antibody.
[0013]
The ISOLATED ANTIBODY according to any one of claims 1 to 11, characterized in that said antibody is a humanized antibody.
[0014]
14. THE ISOLATED ANTIBODY OR IMMUNOREATIVE FRAGMENT THEREOF, according to claim 9, characterized in that said antibody comprises: (A) a light chain variable domain having the amino acid sequence SEQ ID NO: 89; (B) a chain variable domain heavy having the amino acid sequence SEQ ID NO: 99; and (C) an FC region having the substitutions: L235V, F243L, R292P, Y300L, and P396L; wherein the numbering is in accordance with the Kabat numbering scheme.
[0015]
15. ISOLATED ANTIBODY OR IMMUNOREATIVE FRAGMENT THEREOF, according to any one of claims 1 to 14, characterized in that said antibody or its fragment binds to B7 H3 which is expressed by a cancer cell, and in which said cancer cell is selected to from the group consisting of a bladder cancer cell, a cervical cancer, a colon cancer, a colorectal cancer, a gastric cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a prostate cancer, a kidney cancer, a breast cancer, a head and neck cancer, a skin cancer, a sarcoma, a brain tumor, a brain and spinal cord cancer, an adrenal cancer, a uterine cancer , a neuroblastoma, a small round cell tumor, a peripheral nerve sheath tumor, a bone cancer, a rhabdoid tumor, a lymphoma, a multiple myeloma, a leukemia, a neuroendocrine tumor, and a melanoma.
[0016]
16. NUCLEIC ACID MOLECULE, characterized in that it encodes the isolated antibody, as defined in any one of claims 1 to 15, or the immunoreactive fragment, as defined in any one of claims 1 to 6.
[0017]
17. PHARMACEUTICAL COMPOSITION, characterized in that it comprises (i) a therapeutically effective amount of the isolated antibody or immunoreactive fragment, as defined in any one of claims 1 to 15, and (ii) a pharmaceutically acceptable vehicle.
[0018]
18. PHARMACEUTICAL COMPOSITION according to claim 17, characterized in that it additionally comprises an anticancer agent.
[0019]
19. PHARMACEUTICAL COMPOSITION according to claim 18, characterized in that said anticancer agent is a chemotherapeutic agent, a radiation therapeutic agent, a hormonal therapeutic agent, a toxin or an immunotherapeutic agent.
[0020]
20. PHARMACEUTICAL COMPOSITION according to claim 19, characterized in that said anticancer agent is a toxin selected from the group consisting of: a taxane, a maytansinoid, an auristatin, a calicheamicin, an anthracycline, an analogue CC-1065, docetaxel, a cathepsin, ricin, gelonin, PSEUDOMONAS exotoxin, diphtheria toxin, RNase and a toxic radioisotope.
[0021]
21. USE OF THE ISOLATED ANTIBODY or immunoreactive fragment, as defined in any one of claims 1 to 15, or of the pharmaceutical composition, as defined in any one of claims 17 to 20, in an EX VIVO method of cancer diagnosis, characterized by said isolated antibody, said immunoreactive fragment or said diabody is detectably labeled.
[0022]
22. USE according to claim 21, characterized in that said cancer is selected from the group consisting of an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft tissue sarcoma, an astrocytic tumor, bladder cancer, bone cancer, brain or spinal cord cancer, a metastatic brain tumor, a breast cancer, carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobic renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer, a small round cell desmoplastic tumor, an ependynoma, an Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a cancer of head and neck, hepatocellular carcinoma, an islet cell tumor, a Kaposi's sarcoma, a kidney cancer, a leukemia, a liposarcoma/malignant lipomatous tumor, a liver cancer. cattle, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasm, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary carcinoma of the thyroid, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a metastatic renal cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma , skin cancer, soft tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.
[0023]
23. USE OF THE ISOLATED ANTIBODY or the immunoreactive fragment as defined in any one of claims 1 to 15, or of the pharmaceutical composition as defined in any one of claims 17 to 20, characterized in that it is in the preparation of a drug for the treatment of cancer in a patient.
[0024]
24. USE according to claim 23, characterized in that said cancer is selected from the group consisting of an adrenal gland tumor, an AIDS-associated cancer, an alveolar soft tissue sarcoma, an astrocytic tumor, bladder cancer, bone cancer, brain cancer or spinal cord, a metastatic brain tumor, a breast cancer, carotid body tumors, a cervical cancer, a chondrosarcoma, a chordoma, a chromophobic renal cell carcinoma, a clear cell carcinoma, a colon cancer, a colorectal cancer , a small round cell desmoplastic tumor, an ependynoma, an Ewing's tumor, an extraskeletal myxoid chondrosarcoma, a gallbladder or bile duct cancer, gastric cancer, a gestational trophoblastic disease, a germ cell tumor, a head cancer and neck, hepatocellular carcinoma, an islet cell tumor, a Kaposi's sarcoma, a kidney cancer, a leukemia, a liposarcoma/malignant lipomatous tumor, a liver cancer ado, a lymphoma, a lung cancer, a medulloblastoma, a melanoma, a meningioma, a multiple endocrine neoplasm, a multiple myeloma, a myelodysplastic syndrome, a neuroblastoma, neuroendocrine tumors, an ovarian cancer, a pancreatic cancer, a papillary carcinoma of the thyroid, a parathyroid tumor, a pediatric cancer, a peripheral nerve sheath tumor, a pheochromocytoma, a pituitary tumor, a prostate cancer, a posterior uveal melanoma, a metastatic renal cancer, a rhabdoid tumor, a rhabdomyosarcoma, a sarcoma , skin cancer, soft tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

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法律状态:
2017-09-12| B15I| Others concerning applications: loss of priority|

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2017-11-14| B12F| Other appeals [chapter 12.6 patent gazette]|

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2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

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2019-07-16| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-08-04| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-09-29| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-03-30| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-06-08| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-08-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 01/03/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

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US31069210P| true| 2010-03-04|2010-03-04|

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US31069510P| true| 2010-03-04|2010-03-04|

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US61/310,692|2010-03-04|

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